Epstein-Barr Virus Isolates Research Articles

Epstein-Barr virus isolates with the major HLA B35.01-restricted cytotoxic T lymphocyte epitope are prevalent in a highly B35.01-positive African population.

An influence of cytotoxic T lymphocyte (CTL) response over Epstein-Barr virus (EBV) evolution was first suggested by the finding that virus isolates from highly HLA-A11-positive Oriental populations were specifically mutated in two immunodominant A11-restricted CTL epitopes. Here we turn to a second HLA allele, B35.01 and show that B35.01-restricted CTL responses in Caucasian donors reproducibly map to a single peptide epitope, YPLHEQHGM, representing residues 458-466 of the type 1 EBV nuclear antigen 3A protein (B95.8 strain). In this case, however, most EBV isolates from a highly B35.01-positive population (in The Gambia) either retained the CTL epitope sequence or carried a mutation (P-->S at position 2) which conserved antigenicity; changes leading to reduced antigenicity (Y-->N at position 1) were found in only a minority of cases. Furthermore, CTL recognizing the YPLHEQHGM epitope could be reactivated from the blood of some B35.01-positive Gambian donors by in vitro stimulation with the synthetic peptide, indicating that epitope-specific immunity does exist in this population. Possible differences between the A11-based and B35.01-based studies are discussed.

Epstein--Barr virus types 1 and 2 have nearly identical LMP-1 transforming genes

Although variation in the gene encoding the Epstein-Barr virus (EBV) nuclear protein 2 accounts for much of the difference in the transforming activities of the two EBV strain types, divergence in the principal lymphocyte growth-altering gene, LMP-1, has not been previously evaluated. We have now determined the nucleotide sequence of the LMP-1 gene of a type 2 isolate of EBV, AG876. Surprisingly, the AG876 LMP-1 protein is 93% identical to the prototype type 1 EBV strain B95-8, and this is well within the range of variability previously noted among type 1 EBV LMP-1 genes.

Sequence variation in the Epstein-Barr virus latent membrane protein 1.

The sequence of the latent membrane protein 1 (LMP-1) gene was analysed in Epstein-Barr virus (EBV) isolates from specific regions representing both type 1 and type 2 EBV. A predominant strain marked by an XhoI restriction enzyme polymorphism (REP) within the LMP-1 gene has been identified in type 1 EBV in nasopharyngeal carcinoma (NPC) from Southern China. This polymorphism was also present in type 2 EBV in NPC from Alaska. In this study, the sequence of the LMP-1 gene was determined in these samples representing type 1 and type 2 EBV and was compared with the prototype lymphoid strains. Consistent nucleotide variation in the amino terminus of LMP-1 was identified in strains marked by the XhoI REP. These changes were present in both EBV type 1 and type 2 strains. Three types of sequence variation were detected in the carboxy terminus of LMP-1. The LMP-1 sequences differed in the number of an 11 amino acid repeat element. In the prototype EBV type 1 (B95-8) sequence and in the type 1 Raji and type 2 HR-1 strains, the third repeat element contained an insertion of 5 amino acids that were also the first five unique amino acids after the last partial repeat element. The third variation was a deletion of amino acids 343 to 352 of the B95-8 LMP-1. This deletion was detected in the type 1 Chinese EBV strains, but was not detected in the type 2 Alaskan strains although the Chinese and Alaskan strains have nearly identical amino acid changes at the amino terminus. Numerous other amino acid changes were detected in the carboxy terminus which did not cosegregate with either EBV type, amino acid changes in the amino terminus, or specific geographic regions. These data indicate that EBV strains can be distinguished by sequence differences within LMP-1 and that unlike the divergence between type 1 and type 2 EBV in Epstein-Barr nuclear antigen sequences, different EBV types are nearly identical in LMP-1 sequence.

Isolation of Epstein-Barr virus (EBV)-negative cell clones from the EBV-positive Burkitt's lymphoma (BL) line Akata: malignant phenotypes of BL cells are dependent on EBV

During cultivation of the Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) line Akata, it was noted that EBV DNA is lost from some of the cells. Isolation of EBV-positive and EBV-negative clones with the same origin made it possible to examine the effects of EBV in BL cells. The results indicate that malignant phenotypes of BL, such as growth in low serum, anchorage-independent growth in soft agar, and tumorigenicity in nude mice, are dependent on the presence of EBV genomes and underline the oncogenic function of EBV in human cancer.

Epstein-Barr Virus BHRF1 Protein Protects against Cell Death Induced by DNA-Damaging Agents and Heterologous Viral Infection

The BHRF1 open reading frame of Epstein-Barr virus (EBV) codes for a 191-amino-acid protein that is expressed at high levels in productively infected cells and in certain latently infected cells. The BHRF1-coding sequences are conserved among all EBV isolates examined. BHRF1 shares a distant colinear amino acid sequence homology with the protein coded by the Bcl-2 protooncogene which suppresses apoptotic cell death induced by multiple stimuli. We have established Chinese hamster ovary cell lines which constitutively express the BHRF1 protein and show that this viral protein can protect against apoptosis induced by the DNA-damaging agents cisplatin, etoposide, and mitomycin C. The BHRF1 protein also strongly suppresses DNA fragmentation induced by an adenovirus E1B 19K deletion mutant. These results suggest that the BHRF1 protein is functionally similar to Bcl-2 and adenovirus E1B 19-kDa proteins. Since BHRF1 efficiently suppresses apoptosis induced by anti-cancer agents, its expression may have important implications for the chemotherapy of EBV-induced malignancies.

Precipitation of the Epstein-Barr virus protein EBNA 2 by an EBNA 3c-specific monoclonal antibody.

Two monoclonal antibodies, E3cD8 and E3cA10, were generated to the EBNA 3c nuclear protein from the B95.8 isolate of Epstein-Barr virus (EBV). Both antibodies efficiently precipitate EBNA 3c from B95.8-transformed lymphoblastoid cell lines, and E3cA10 also detects EBNA 3c on Western blots. Whereas E3cD8 reacts with all 11 Type-1 isolates of EBV tested, and E3cA10 reacts with 14 of 17 Type-1 isolates, neither antibody detects the EBNA 3c protein encoded by Type-2 isolates. E3cD8 recognizes a peptide sequence (PA/PPQAPYQGY) in a repeat region of the B95.8 EBNA 3c coding sequence which is not present in the prototype Type-2 AG876 sequence. The E3cA10 antibody epitope has been mapped to the minimal five amino acid B95.8 peptide, WAPSV, which has an alanine to valine substitution in the AG876 virus isolate. This substitution was also found in three Type-1 EBV isolates that expressed EBNA 3c proteins not detected by E3cA10. In immunoprecipitation studies E3cA10 additionally coprecipitated the EBNA 2 protein from Type-1 isolates of EBV. The possibility of a direct interaction between EBNA 2 and EBNA 3c was ruled out by the demonstration that the antibody precipitated EBNA 2 from the Raji cell line which carries a virus with a deleted EBNA 3c gene. Since the WAPSV epitope identified in EBNA 3c is not present in EBNA 2, and no EBNA 2 linear peptide reactivity was detected in ELISA, it seems likely that E3cA10 recognizes a conformational epitope on EBNA 2. However, from the present data we cannot exclude the possibility that the antibody reacts with a cellular protein that physically associates with EBNA 2.

T cell responses and virus evolution: loss of HLA A11-restricted CTL epitopes in Epstein-Barr virus isolates from highly A11-positive populations by selective mutation of anchor residues.

Epstein-Barr virus (EBV) is a B lymphotropic herpesvirus of humans that elicits strong HLA class I-restricted cytotoxic T lymphocyte (CTL) responses. An influence of such responses on virus evolution was first suggested by our finding that EBV isolates from the highly HLA A11-positive Papua New Guinea (PNG) population carried a lys-thr mutation at residue 424 of the nuclear antigen EBV-encoded nuclear antigen (EBNA4) that destroyed the immunodominant target epitope for A11-restricted CTL recognition. Here we turn to a much larger population, Southern Chinese, where the A11 allele is again present in over 50% of the individuals. Each of 23 EBV isolates analyzed from this population were also mutated in the EBNA4 416-424 epitope, the mutations selectively involving one of the two anchor residues in positions 2 (417 val-leu) or 9 (424 lys-asp, -arg or -thr) that are critical for A11-peptide interaction. The majority of the Chinese isolates and all 10 PNG isolates also carried mutations affecting positions 1 and 2 of the next most immunodominant A11-restricted epitope, EBNA4 residues 399-408. These changes clearly affected antigenicity since A11-positive lymphoblastoid cell lines (LCLs) carrying these mutant EBV strains were not recognized by A11-restricted CTLs raised against the prototype B95.8 virus. Furthermore, Chinese donors naturally infected with these mutant viruses did not mount detectable A11-restricted CTL responses on in vitro stimulation with autologous LCL cells carrying either the B95.8 or their endogenous EBV strain. In two different highly A11-positive populations, therefore, immune pressure appears to have selected for resident EBV strains lacking immunodominant A11-restricted CTL epitopes.

HLA A2.1-restricted cytotoxic T cells recognizing a range of Epstein-Barr virus isolates through a defined epitope in latent membrane protein LMP2

Cytotoxic T-lymphocyte (CTL) responses induced by persistent Epstein-Barr virus (EBV) infection in normal B-lymphoid tissues could potentially be directed against EBV-positive malignancies if expression of the relevant viral target proteins is maintained in tumor cells. For malignancies such as nasopharyngeal carcinoma and Hodgkin's disease, this will require CTL targeting against the nuclear antigen EBNA1 or the latent membrane proteins LMP1 and LMP2. Here we analyze in detail a B95.8 EBV-reactivated CTL response which is specific for LMP2 and restricted through a common HLA allele, A2.1. We found that in vitro-reactivated CTL preparations from several A2.1-positive virus-immune donors contained detectable reactivity against A2.1-bearing target cells expressing either LMP2A or the smaller LMP2B protein from recombinant vaccinia virus vectors. Peptide sensitization experiments then mapped the A2.1-restricted response to a single epitope, the nonamer CLGGLLTMV (LMP2A residues 426 to 434), whose sequence accords well with the proposed peptide binding motif for A2.1. Most Caucasian and African virus isolates (whether of type 1 or type 2) were identical in sequence to B95.8 across this LMP2 epitope region, although 2 of 12 such isolates encoded a Leu-->Ile change at epitope position 6. In contrast, most Southeast Asian and New Guinean isolates (whether of type 1 or type 2) constituted a different virus group with a Cys-->Ser mutation at epitope position 1. CTLs raised against the B95.8-encoded epitope were nevertheless able to recognize these variant epitope sequences in the context of A2.1 whether they were provided exogenously as synthetic peptides or generated endogenously in B cells transformed with the variant viruses. A CTL response of this kind could have therapeutic potential in that it is directed against a protein expressed in many EBV-positive malignancies, is reactive across a range of virus isolates, and is restricted through a relatively common HLA allele.

Sequence variation of cytotoxic T cell epitopes in different isolates of Epstein-Barr virus.

Previous results have identified two distinct cytotoxic T lymphocyte (CTL) epitopes encoded by Epstein-Barr virus (EBV), TETA (ORF BLRF3/BERF1 residues 329-353) and EENL (ORF BERF3/BERF4 residues 290-309). Measurement of the specificities of CTL clones (TETA-specific clone 13 and EENL-specific clone 7) directed against these epitopes indicated that the EENL epitope is conserved in all strains of EBV tested while the TETA epitope varied between individual virus strains. Sequencing of the DNA regions encoding these two CTL epitopes in different EBV isolates confirmed these interpretations and demonstrated that different TETA epitope sequences were encoded by B-type EBV strains and by the B95-8 isolate of EBV compared to the other A-type EBV strains. Titration of synthetic variants of the TETA epitope revealed that the epitope encoded by B95-8 was 15-fold less efficient as a T cell epitope than the sequence encoded by other A-type viral strains while the TETA variant encoded by the B-type strains displayed essentially no activity as a T cell epitope.

Epstein-Barr virus infection in allogeneic marrow grafting: lessons for transplant physicians and virologists.

The relationship between Epstein-Barr virus (EBV) and the host is profoundly disturbed by allogeneic bone marrow transplantation (BMT) because EBV resides in the recipient's hematopoietic system, which has to be destroyed in the majority of cases, and in the donor's hematopoietic system, i.e., the marrow graft. We have shown that EBV may be eradicated from some BMT recipients and that the virus may be transferred with the marrow graft. During the immediate post-transplant period oropharyngeal EBV excretion may occur which, by infecting passing B lymphocytes, may act as co-factor for acute graft-versus-host disease and help the virus to survive, despite the temporary depletion of its reservoir. The coexistence of totally different EBV strains in BMT recipients but not in healthy, untransfused controls, suggests that superinfection may by possible in case of immunodeficiency; alternatively, transfer of the virus by the reservoir itself (the B lymphocytes) might be the only effective route for superinfection. The generation of 'variant' strains during viral replication may form the basis of the vast polymorphism between wild-type EBV isolates in the population.

Isolation and sequencing of the Epstein-Barr virus BNLF-1 gene (LMP1) from a Chinese nasopharyngeal carcinoma

The BamHI fragment containing the Epstein-Barr virus (EBV) LMP1 gene was cloned from a genomic library of the nude mouse-propagated Chinese nasopharyngeal carcinoma CAO. The sequence of the LMP1 gene and its promoter and enhancer was determined. The nucleotide sequence of the CAO isolate differed from those of the B95-8 and Raji isolates in the promoter/enhancer region; the amino acid sequence of the protein also differed. Structural differences in the protein were located mainly in the 20 N-terminal residues and the array of repeated amino acids in the C-terminal part of the protein, in which the CAO isolate displays a cluster of seven perfect repeats of 11 amino acids (aa). Three of these repeats have no counterpart in the other virus strains. This, together with two deletions of five and 10 aa in the C-terminal part, yields a protein of 404 aa, compared to 386 aa for B95-8 and Raji. The larger LMP1 protein was detected on immunoblots of tissue samples from the CAO nude mouse tumour, and was also present in EBV-negative B cell lines and immortalized keratinocytes transfected with the cloned gene. A XhoI restriction site in exon 1 of the B95-8 BNLF-1 gene was absent from the CAO EBV isolate, as well as from 36 of 37 Chinese NPC biopsies tested. In contrast, 17 of 19 NPC biopsies of African origin retained this XhoI site.

Persistence of Epstein-Barr virus in Reed-Sternberg cells throughout the course of Hodgkin's disease.

Non-isotopic in situ hybridization employing digoxigenin-labelled DNA probes has been used to localize Epstein-Barr virus (EBV) in 55 cases of Hodgkin's disease (HD). The virus was found in Reed-Sternberg (RS) and mononuclear Hodgkin's cells in nine patients (16 per cent). Further samples taken at different times from three patients also showed the presence of EBV in the malignant cell population. Estimations of the number of EBV genomes present per cell suggested wide variations between different patients, but relatively constant amounts in different samples from the same patient. These findings are compatible with a stable infection of the neoplastic cells and support the notion that EBV may play a role in the development of HD in these patients. We also found evidence for the presence of EBV in a small percentage of non-neoplastic cells in 8 of the 55 samples. This suggests that isolation of EBV from HD tissue does not always signify a pathogenetic role for the virus. Furthermore, it is apparent that a high percentage of HD tissues do not contain demonstrable EBV, and the virus is therefore unlikely to be a causative agent for all cases of HD.

Identification of type A and B isolates of Epstein-Barr virus by polymerase chain reaction

A method is described for the identification of type A and type B isolates of Epstein-Barr virus (EBV) by means of the polymerase chain reaction. The use of three pairs of primers specific for genomic sequences coding for the two forms of EBV nuclear antigen (EBNA), 2A and 2B, and for a DNA sequence from the BamZ/ BamR region allows the reliable and rapid detection of type A and B viruses in as little as 1000 EBV positive cells.

Modulation of cellular gene expression in B lymphoma cells following in vitro infection by Epstein‐Barr virus (EBV)

In vitro infection of EBV-negative lymphoma cell lines with immortalizing strains of Epstein-Barr virus induces the cell-surface expression of B-cell markers, such as the EBV receptor/CR2 (CD21) and the CD23 antigen. The non-immortalizing EBV variant, P3HRI, which carries a deletion encompassing the EBV nuclear antigen 2 (EBNA2) gene, fails to induce any such expression. We show here that the EBV-mediated up-regulation of cell-surface expression of these molecules is associated with an increased level of the specific steady-state RNA corresponding to these 2 genes. These results suggest that the role of EBNA2 in B-cell growth and immortalization may be related to its role in transactivation of cellular genes. In order to identify other cellular genes whose expression may be modulated by EBV, we analyzed the level of transcription of a set of genes possibly involved in Burkitt's lymphoma pathogenesis. The level of the c-myc oncogene transcript was not significantly affected by in vitro EBV infection. The c-fgr oncogene, thought to be specifically activated in EBV-infected cells, was found to be expressed in some EBV-negative lymphoma cells and also to be activated by both non-immortalizing and immortalizing strains of EBV. The expression of vimentin, the major 56-kDa polypeptide of mesenchymal cell intermediate filaments, was altered by all EBV isolates, in either a negative or a positive way, depending on the cell line. Expression of lymphocyte-function-associated antigens, LFA-1 alpha/beta (CD11 a/18) and LFA-3 (CD58), involved in intercellular adhesion and the T-cytotoxic pathway, were differentially regulated by EBV; a crucial observation was the activation by immortalizing EBV isolates of LFA-1 beta chain (CD18) and of LFA-3 (CD58). The EBV-and possibly EBNA2-associated modulation of cellular genes, such as CR2 (CD21), CD23 and LFAs, probably represents key events for EBV-induced B-cell proliferation, and also for in vivo immune control of EBV-infected B cells.

Oligonucleotides for polymerase chain reaction amplification and hybridization detection of Epstein-Barr virus DNA in clinical specimens

We designed synthetic oligonucleotide primers and hybridization probe for use in polymerase chain reaction (PCR) amplification and hybridization detection of Epstein-Barr virus (EBV) nucleic acid sequences. Primer sequences were chosen from the coding region for the Epstein-Barr virus nuclear antigen-1 (EBNA-1). PCR amplification and hybridization with these oligonucleotides was carried out on standard laboratory cell lines including African Burkitt's lymphoma and infectious mononucleosis derived cell lines, as well as cell lines recently established from clinical EBV isolates from bone marrow transplant recipients. All EBV cell lines tested were positive. No false-positives were detected with uninfected cell lines, human placental DNA or with other viruses. The sensitivity of the detection procedure was such that four copies of the EBV genome could consistently be detected in a background of 1 μg of placental DNA. EBV was detected in DNA extracts from the peripheral blood mononuclear cells of two patients with infectious mononucleosis and one patient with viral-associated hemophagocytic syndrome. Three of 18 EBV seropositive patients without known ongoing EBV-associated illness undergoing ambulatory surgery also had EBV detected in DNA extracts from their peripheral blood mononuclear cells. EBV was detected in DNA extracts from lymphoma tissue from two patients with post-transplant lymphomas and two AIDS patients with primary CNS lymphomas. EBV was not detected in 12 B-cell lymphoma specimens from patients without history of immunocompromise. DNA extracts from formalin-fixed paraffin-embedded Hodgkin's tissues previously shown to be EBV positive by Southern blot were also demonstrated to be EBV positive by PCR. Thus, with the oligonucleotides described, PCR is applicable to the detection of EBV in a spectrum of clinical isolates. The broad specificity of these oligonucleotides for all strains of EBV tested is probably a function of the highly conserved sequence of the EBNA-1 DNA binding domain.

Epstein-Barr virus types 1 and 2 differ in their EBNA-3A, EBNA-3B, and EBNA-3C genes

The two Epstein-Barr virus (EBV) types, EBV-1 and EBV-2, are known to differ in their EBNA-2 genes, which are 64 and 53% identical in their nucleotide and predicted amino acid sequences, respectively. Restriction endonuclease maps and serologic analyses detect few other differences between EBV-1 and EBV-2 except in the EBNA-3 gene family. We determined the DNA sequence of the AG876 EBV-2 EBNA-3 coding region and have compared it with known B95-8 EBV-1 EBNA-3 sequences to delineate the extent of divergence between EBV-1 and EBV-2 isolates in their EBNA-3 genes. The B95-8 and AG876 EBV isolates had nucleotide and amino acid identity levels of 90 and 84%, 88 and 80%, and 81 and 72% for the EBNA-3A, -3B, and -3C genes, respectively. In contrast, nucleotide sequence identity in the noncoding DNA adjacent to the B95-8 and AG876 EBNA-3 open reading frames was 96%. We used the polymerase chain reaction to demonstrate that five additional EBV-1 isolates and six additional EBV-2 isolates have the type-specific differences in their EBNA-3 genes predicted from the B95-8 or AG876 sequences. Thus, EBV-1 and EBV-2 are two distinct wild-type EBV strains that have significantly diverged at four genetic loci and have maintained type-characteristic differences at each locus. The delineation of these sequence differences between EBV-1 and EBV-2 is essential to ongoing molecular dissection of the biologic properties of EBV and of the human immune response to EBV infection. The application of these data to the delineation of epitopes recognized in the EBV-immune T-cell response is also discussed.

Differences in the ability of cells to fuse are mediated by strains of epstein‐barr virus

Epstein-Barr virus (EBV) preparations from both NPC-KT cells (NPC-EBV) and P3HR-1 cells (HR-1-EBV) can induce cell fusion between EBV receptor (EBVR)-positive Raji cells and EBVR-negative cells, but other strains of EBV cannot induce cell fusion. The effect of these two EBV isolates on ability of cells to fuse has been studied to determine if there are differences in the biological properties of the different EBV isolates, particularly the isolates obtained from nasopharyngeal carcinoma such as NPC-EBV. The frequency of cell fusion between NPC-EBV-superinfected Raji cells and EBVR-negative epithelial cells (Ad-AH) was increased more than 30-fold in the presence of medium containing 1% dimethylsulfoxide (DMSO). However, the frequency of cell fusion between HR-1-EBV-superinfected Raji cells and Ad-AH cells was unaffected under the same conditions. The data show that differences in the ability of cells to fuse are induced by variants of EBV in response to DMSO. These differences may be important in elucidating the different biological properties of EBV isolates and might have implications for the pathophysiology of EBV-associated illness.

Predilection of a nasopharyngeal carcinoma‐derived isolate of epstein‐barr virus for infection of specific subsets of B lymphocytes

It is important to know whether there are variants of Epstein-Barr virus (EBV) with biological properties that are different from the prototype viruses that have been studied in detail, such as P3HR-1 and B95-8. We have studied an EBV isolate derived from a nasopharyngeal carcinoma (NPC) tumor, designated NPC-EBV. We have examined the target B lymphocytes infected and growth-transformed with NPC-EBV as compared with two common EBV isolates, B95-8 and AG876 EBV, for stage of maturation using antibodies to several immunoglobulin chains. Typing of the NPC-EBV transformed lymphoblastoid cell lines revealed the predilection of the NPC-EBV isolate to infect immature B lymphocytes. This was not the case for the B95-8 and AG876 isolates. The reason for the predilection of NPC-EBV for immature B lymphocytes remains to be explored further. However, these results may be important in understanding the pathophysiology of EBV-associated diseases.

Distinction between Epstein-Barr virus type A (EBNA 2A) and type B (EBNA 2B) isolates extends to the EBNA 3 family of nuclear proteins

The Epstein-Barr virus (EBV) nuclear antigens EBNA 3a, 3b, and 3c have recently been mapped to adjacent reading frames in the BamHI L and E fragments of the B95.8 EBV genome. We studied by immunoblotting the expression of the family of EBNA 3 proteins in a panel of 20 EBV-transformed lymphoblastoid cell lines (LCLs) carrying either type A (EBNA 2A-encoding) or type B (EBNA 2B-encoding) virus isolates. Certain human sera from donors naturally infected with type A isolates detected the EBNA 3a, 3b, and 3c proteins in all type A virus-transformed LCLs (with a single exception in which EBNA 3b was not detected) but detected only EBNA 3a in LCLs carrying type B isolates. These results were confirmed with human and murine antibodies with specific reactivity against sequences of the type A EBNA 3a, 3b, or 3c expressed in bacterial fusion proteins. Conversely, selected human sera from donors naturally infected with type B strains of EBV identified the EBNA 3a encoded by both types of isolates plus two novel EBNAs present only in type B, and not in type A, virus-transformed LCLs; these novel proteins appear to be the type B homologs of EBNA 3b and 3c. The distinction between type A and type B EBV isolates therefore extends beyond the EBNA 2 gene to the EBNA 3 family of proteins. This has important implications with respect to the evolutionary origin of these two EBV types and also places in a new light recent studies which identified differences between type A and type B transformants in terms of growth phenotype (A. B. Rickinson, L. S. Young, and M. Rowe, J. Virol. 61:1310-1317, 1987) and of detection by EBV-specific cytotoxic T cells (D. J. Moss, I. S. Misko, S. R. Burrows, K. Burman, R. McCarthy, and T. B. Sculley, Nature [London] 331:719-721, 1988).

Gene Mapping of an Epstein-Barr Virus Isolate Obtained from a Nasopharyngeal Carcinoma

Gene mapping of an Epstein-Barr virus (EBV) isolate derived from a nasopharyngeal carcinoma (NPC), designated NPC-EBV, has been performed. This isolate was rescued from an NPC epithelial hybrid cell line (NPC-KT), and used to transform cotton-top tamarin lymphocytes which, along with the epithelial NPC-KT cells, were used in the mapping studies. Using the BamHI and EcoRI restriction enzymes, we found that the NPC-EBV isolate did not contain the deletions observed in the genomes of the prototype HR-1 and B95-8 isolates, and may represent a 'more complete' virus genome. Polymorphism was observed in the BamHI-X, T, H, and L regions as compared to the HR-1 and B95-8 isolates. The NPC-EBV DNA in the epithelial NPC-KT cells was compared to the NPC-EBV genome in three different cotton-top tamarin lymphoblastoid cell lines transformed with the isolate. The restriction patterns were the same with one exception; there were differences in the size of the BamHI N-J het regions. This finding is consistent with the idea that the BamHI N-J het region may be the switch point of EBV DNA from the linear to the episomal form. The NPC-EBV is the first NPC-derived isolate to be obtained from epithelial cells in vivo and maintained in epithelial cells in vitro, and will be useful for studying biological variability of EBV.