Possible involvement in oncogenesis of a single base mutation in an internal ribosome entry site of Epstein-Barr nuclear antigen 1 mRNA

It has been reported recently that the U leader exon located within the 5' untranslated region of Epstein-Barr nuclear antigen 1 (EBNA1) gene contains an internal ribosome entry site (IRES) element. Sequence analysis of the U leader exon was undertaken in samples from 19 patients with infectious mononucleosis and 19 patients with lethal lymphoproliferative diseases and in 15 spontaneously established lymphoblastoid cell lines. The sequence was conserved except for a single base substitution (T-C) at position 67,585. Although the mutation was detected in only one case of infectious mononucleosis, it was found in more than half of the lethal lymphoproliferative diseases and all lymphoblastoid cell lines. The results suggest that a mutation in the IRES element affects EBNA1 gene expression at the translational level and provides Epstein-Barr virus (EBV)-infected cells with a growth advantage, leading to immortalization of cells in vitro and to the development of lethal lymphoproliferative diseases in vivo.     

EBNA1 expression in a lung transplant recipient with hypocomplementemic urticarial vasculitis syndrome

This article describes a transplant recipient with underlying hypocomplementemic urticarial vasculitis syndrome who expressed persistently Epstein-Barr virus nuclear antigen 1 (EBNA1) in peripheral blood. The patient received a bilateral lung transplant and was subsequently followed with monitoring of EBV expression in peripheral blood. Evaluation of viral expression in peripheral blood, serum, and graft tissue was performed with RT-PCR, Q-PCR, indirect immunofluorescence, anti-peptide assays, and in situ hybridization; samples were collected at various time-points up to 91 days post-transplantation. The patient expressed EBNA1 in 8/10 (80%) of the peripheral blood samples tested during the post-transplantation period, and interestingly, even including the day of transplantation. After analyses of indicative EBV mRNA, EBNA1 expression was found mainly to be Qp-initiated EBNA1, known to be important for EBV maintenance. Anti-EBNA1 epitope mapping showed significantly higher and broader antibody responses to EBNA1 epitopes pre-transplantation when compared to normal controls and a matched lung transplant control. Post-transplantation this response was largely diminished but there were still epitopes significantly higher than controls. Our results show the presence of EBV-positive proliferating cells before onset of intensive immunosuppressive treatment. Although no previous connection between EBV and hypocomplementemic urticarial vasculitis syndrome has been reported, it is tempting to speculate that the continuous EBNA1 expression is not caused by immunosuppression or post-transplant lymphoproliferative disease, but may be a factor involved in the etiology of the autoimmune disease.     

Prospective Follow-up of Epstein-Barr Virus Load in Adult Kidney Transplant Recipients by Semiquantitative Polymerase Chain Reaction in Blood and Saliva Samples

The aim of the study presented here was to set up and standardize a semiquantitative polymerase chain reaction method for monitoring Epstein-Barr virus (EBV) DNA levels in blood and saliva samples from transplant recipients and to determine the value of these levels as an early marker for the development of post-transplant lymphoproliferative disorders. EBV DNA load was prospectively measured in 53 adult kidney transplant recipients. Results were correlated with clinical features and degree of immunosuppression. Healthy blood donors and patients with infectious mononucleosis were used as controls. Levels higher than 500 EBV DNA copies/75,000 peripheral blood mononuclear cells were found in all patients with infectious mononucleosis and in all patients with post-transplant lymphoproliferative disorder but in only 7.5% of transplant recipients without that complication. Electronic Publication     

A peptide-based inhibitor for prevention of B cell hyperproliferation induced by Epstein-Barr virus

Epstein-Barr virus (EBV) infects and transforms resting B lymphocytes in vitro. The virus can also cause B cell lymphomas in immunosuppressed humans. Indeed, EBV-mediated post-transplant lymphoproliferative disease causes significant complications in transplant recipients, including loss of the transplanted organ and even death. The limited treatment options include, nonspecific targeting of B cell surface antigens with monoclonal antibodies or withdrawal of immunosuppression. These therapies fail in approximately 50% of patients. Clearly, treatments that specifically target EBV-infected cells are desirable. The EBV antigen EBNA3C regulates cell cycle by targeting critical cellular complexes such as cyclin A/cdk2, SCF(Skp2), and Rb. Here, we use a 20-amino-acid EBNA3C-derived peptide, fused to an HIV TAT tag for efficient delivery, to disrupt cell cycle regulation by EBNA3C. The peptide inhibited hyperproliferation of EBV-infected B cell lines and reduced in vitro immortalization of primary B lymphocytes by EBV. Importantly, the peptide inhibited lymphoblastoid outgrowth from the blood of an EBV-positive transplant patient in vitro.     

Clinical characteristics,outcome and the role of viral load in nontransplant patients with Epstein--Barr viraemia

Epstein--Barr virus (EBV) is important in the development of post-transplant lymphoproliferative disease in allogeneic stem cell and solid organ transplant recipients. We have studied the clinical significance of EBV DNAaemia among nontransplant patients in a tertiary referral hospital. We retrospectively reviewed the medical records for main diagnosis, outcome, immunosuppressive/cytotoxic chemotherapy and other opportunistic infections of the patients who were found -positive in quantitative real-time PCR assay for EBV (EBV-qPCR) between the years 2000 and 2007. Allogeneic stem cell and solid organ transplant recipients were excluded, and all patients in nonsurgical adult wards were included. Altogether, 62 patients had at least one plasma sample -positive with an EBV-qPCR. Fifteen were immunocompetent, most had primary EBV infection, and the outcome was good. On the other hand, 36 had malignant disease, seven had HIV infection and seven had immunosuppressive conditions of an other aetiology. All but one of the malignancies were of lymphoid origin, and most of these patients had a history of multiple cytotoxic treatments. Immunosuppressed patients had higher viral loads. EBV viraemia is associated with severe immunosuppression and lymphoid malignancies.     

Determining virological, serological and immunological parameters of EBV infection in the development of PTLD

Post-transplant lymphoproliferative disease (PTLD) in Epstein-Barr virus (EBV) seronegative solid organ transplant recipients remains a significant problem, particularly in the first year post-transplant. Immune monitoring of a cohort of high-risk patients indicated that four EBV seronegative transplant recipients developed early-onset PTLD prior to evidence of an EBV humoral response. EBV status has been classically defined serologically, however these patients demonstrated multiple parameters of EBV infection, including the generation of EBV-specific CTL, outgrowth of spontaneous lymphoblastoid cell lines, and elevated EBV DNA levels, despite the absence of a classic EBV antibody response. As EBV serology is influenced by both immunosuppression and cytomegalovirus immunoglobulin treatment, both the EBV-specific CTL response and elevated EBV levels are more reliable indicators of EBV infection post-transplant.     

Quantitative Epstein-Barr virus (EBV) serology in lung transplant recipients with primary EBV infection and/or post-transplant lymphoproliferative disease

The Epstein-Barr virus (EBV)-specific antibody response was studied in lung transplant patients to assess their value in the diagnosis and prognosis of post-transplant lymphoproliferative disease. Recently developed synthetic peptides representing Epstein-Barr nuclear antigen-1 (EBNA-1), diffuse early antigen (EA(D)), and virus capsid antigen (VCA) were studied in a semiquantitative enzyme-linked immunosorbent assay (ELISA) to study antibody patterns in 12 seronegative lung transplant patients, of whom four developed a post-transplant lymphoproliferative disease, and seven seropositive lung transplant patients, all of whom developed a post-transplant lymphoproliferative disease. Immunoblot technique was used as a control. All 12 EBV-seronegative patients had a very limited antibody response that was restricted mainly to VCA antibodies. EA(D) antibodies became detectable in only two patients. Antibody response never preceded clinical diagnosis of post-transplant lymphoproliferative disease in the four EBV-seronegative patients who developed post-transplant lymphoproliferative disease. In the seven seropositive lung transplant patients with post-transplant lymphoproliferative disease, we found a rise in antibody titer in only two patients. Immunoblot analysis confirmed the serological results. In conclusion, EBV-specific antibody patterns after lung transplantation are highly restricted and variable and of limited value for the diagnosis or prognosis of post-transplant lymphoproliferative disease.     

Pediatric solid-organ transplant recipients carry chronic loads of Epstein-Barr virus exclusively in the immunoglobulin D-negative B-cell compartment

Solid-organ transplant recipients are at risk for development of lymphoproliferative diseases. The purpose of this study was to examine the distribution of Epstein-Barr virus (EBV) load in the peripheral blood of pediatric transplant recipients who had become chronic viral load carriers (>8 copies/10(5) lymphocytes for >2 months). A total of 19 patients with viral loads ranging from 20 to 5,000 viral genome copies/10(5) lymphocytes were studied. Ten patients had no previous diagnosis of posttransplant lymphoproliferative disease (PT-LPD), while nine had recovered from a diagnosed case of PT-LPD. No portion of the peripheral blood viral load was detected in the cell-free plasma fraction. Viral DNA was found in a population of cells characterized as CD19(hi) and immunoglobulin D negative, a phenotype that is consistent with the virus being carried exclusively in the memory B-cell compartment of the peripheral blood. There was no difference in the compartmentalization based upon either the level of the viral load or the past diagnosis of an episode of PT-LPD. These results have implications for the design of tests to detect EBV infection and for the interpretation and use of positive EBV PCR assays in the management of transplant recipients.     

Characteristics of viral protein expression by Epstein-Barr virus-infected B cells in peripheral blood of patients with infectious mononucleosis.

The frequency of Epstein-Barr virus (EBV) antigen-positive B cells in the peripheral blood of patients with infectious mononucleosis compared with that for latently EBV-infected individuals was examined by immunocytochemistry. B cells positive for Epstein-Barr nuclear antigen (EBNA) 1, EBNA2, and latent membrane protein were frequently found in all peripheral B lymphocyte preparations from 25 patients suffering for 3 to 28 days from infectious mononucleosis by using monoclonal antibodies and the alkaline phosphatase anti-alkaline technique. There was a significant decrease in the number of positive B cells during the course of disease. EBNA1-positive B cells were detected in 0.01 to 2.5% of total B cells (median, 0.8%), EBNA2-positive B cells were detected in 0.01 to 4.5% of total B cells (median, 0.9%), and latent membrane protein-positive B cells were detected in 0.01 to 1.8% of total B cells (median, 0.5%), depending on the duration of clinical signs. In contrast, we did not find any EBNA1- or EBNA2-positive B cells in 2 x 10(6) peripheral blood B lymphocytes of 10 latently EBV-infected individuals, whereas aliquots of the same cell preparations were EBV DNA positive by a PCR assay. Therefore, it appears to be possible to detect infectious mononucleosis by immunocytochemical determination of latent EBV products, which might be of relevance for the diagnosis of EBV reactivations in immunosuppressed patients.     

Epstein-Barr virus infection in paediatric liver transplant recipients: detection of the virus in post-transplant tonsillectomy specimens.

AIMS: Post-transplant lymphoproliferative disease (PTLD) is an important and serious complication in transplant patients. Recent studies have suggested that quantitative assessment of Epstein-Barr virus (EBV) infection in transplant patients might help to identify those at risk of developing PTLD. Therefore, tonsils from paediatric liver transplant recipients were studied for evidence of EBV infection. METHODS: Tonsils were studied by in situ hybridisation for the detection of the small EBV encoded nuclear RNAs (EBERs). The phenotype of EBV infected cells was determined by double labelling in situ hybridisation and immunohistochemistry. The expression of viral latent and lytic antigens was determined by immunohistochemistry. Tonsils from patients without known immune defects were studied as controls. RESULTS: Tonsils from transplant patients showed pronounced follicular hyperplasia and minor paracortical hyperplasia. In situ hybridisation revealed variable numbers of EBV infected B cells in the tonsils from transplant patients (range, 2-1000/0.5 cm(2); mean, 434/0.5 cm(2); median, 105/0.5 cm(2)). Lower numbers were detected in the control tonsils (range, 1-200/0.5 cm(2); mean, 47/0.5 cm(2); median, 9/0.5 cm(2)). The latent membrane protein 1 (LMP1) of EBV was not detected and there were only rare cells in two cases showing expression of the EBV encoded nuclear antigen 2 (EBNA2). There was no evidence of lytic infection. None of the patients developed PTLD within a follow up period of up to five years. CONCLUSIONS: These data indicate that tonsillar enlargement in paediatric liver transplant patients does not necessarily imply a diagnosis of PTLD. Furthermore, the presence of increased numbers of EBV infected cells in tonsils from liver transplant recipients by itself does not indicate an increased risk of developing PTLD.     

Monitoring of Epstein-Barr virus load after hematopoietic stem cell transplantation for early intervention in post-transplant lymphoproliferative disease

Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease is a life-threatening complication following hematopoietic stem cell transplantation. A quantitative polymerase chain reaction to evaluate EBV-genome copy numbers based on a nested polymerase chain reaction and an end-point dilution was used. Applying this assay EBV load was prospectively screened weekly in 123 patients after transplantation. The results demonstrate that EBV reactivations with more than 1,000 EBV-genome copies measured in 10(5) peripheral blood mononuclear cells were observed in 31 patients (25.2%). Three patients developed lymphoproliferative disease with extremely high EBV-genome copies in peripheral blood mononuclear cells (>100,000 copies/10(5) cells) and plasma. After combined antiviral and immune therapy two of three patients showed a dramatic decrease of EBV load and survived, while the third patient died of lymphoma. A subclinical EBV reactivation was observed in 24 cases (19.5%) with EBV-genome copies in 10(5) peripheral blood mononuclear cells ranging between 2,500 and mostly 10,000. After reduction of immunosuppression the EBV levels normalized. In four patients, the high copy number of > or =80,000 copies/10(5) peripheral blood mononuclear cells and plasma positivity prompted us to start pre-emptive therapy with rituximab and cidofovir for prevention of lymphoproliferative disease. After drug administration the high EBV load was reduced remarkably. Ninety-two patients (74.8%) who had < or =1,000 copies/10(5) peripheral blood mononuclear cells did not develop EBV-associated lymphoproliferative disease. In conclusion, monitoring of EBV load is a sensitive and useful parameter in the surveillance of EBV reactivation for early intervention in EBV-associated lymphoproliferative disease as well as for follow-up of the efficacy of therapy.     

Epstein-Barr virus gene expression and latent membrane protein 1 gene polymorphism in pediatric liver transplant recipients

Immunosuppressed pediatric transplant recipients are at risk of developing Epstein–Barr virus (EBV)‐associated complications (such as post‐transplant lymphoproliferative disorders). Monitoring of the EBV DNA level in blood alone has a low predictive value for the post‐transplant course of EBV infection and its complications. Therefore, additional prognostic markers are widely sought. The study aim was to analyze EBV gene expression patterns and LMP1 polymorphism in relation to EBV DNA levels in pediatric liver transplant recipients. EBV load measurement, LMP1 variant, and gene expression analysis were performed in collected prospectively multiple blood samples from 30 patients. Several distinct patterns of EBV gene expression were identified: latency 2 (71%), latency 3 (13%), latency 0 (11%), and lytic infection (5%). In most children's multiple blood samples, both EBV gene expression patterns and expression levels of individual EBV genes varied significantly over time. EBV gene expression patterns were not associated with the EBV load. However, the viral load correlated with the LMP1 and LMP2 expression (r = 0.34; P = 0.006, and r = 0.45; P = 0.001, respectively). Two variants of the LMP1 gene were detected, and they were consistent over time in individual patients. A wild type of LMP1 was associated with higher EBV‐DNA loads (P = 0.03). This indicates that EBV infection in immunosuppressed patients is a very dynamic process, but changes in the state of EBV infection do not influence significantly the viral load. The latter, however, can be associated with the activity of LMP1 and LMP2 genes, as well as polymorphism of LMP1. J. Med. Virol. 83:2182–2190, 2011. © 2011 Wiley Periodicals, Inc.     

Expression of Epstein-Barr virus-encoded small RNA (by the EBER-1 gene) in liver specimens from transplant recipients with post-transplantation lymphoproliferative disease.

BACKGROUND. Epstein-Barr virus (EBV)-associated post-transplantation lymphoproliferative disease (PTLD) develops in 1 to 10 percent of transplant recipients, in whom it can be treated by a reduction in the level of immunosuppression. We postulated that the tissue expression of the small RNA transcribed by the EBER-1 gene during latent EBV infection would identify patients at risk for PTLD. METHODS. We studied EBER-1 gene expression in liver specimens obtained from 24 patients 2 days to 22 months before the development of PTLD, using in situ hybridization with an oligonucleotide probe. Control specimens were obtained from 20 recipients of allografts with signs of injury due to organ retrieval, acute graft rejection, or viral hepatitis in whom PTLD had not developed 9 to 71 months after the biopsy. RESULTS. Of the 24 patients with PTLD, 17 (71 percent) had specimens in which 1 to 40 percent of mononuclear cells were positive for the EBER-1 gene. In addition, 10 of these 17 patients (59 percent) had specimens with histopathological changes suggestive of EBV hepatitis. In every case, EBER-1-positive cells were found within the lymphoproliferative lesions identified at autopsy. Only 2 of the 20 controls (10 percent) had specimens with EBER-1-positive cells (P < 0.001), and such cells were rare. CONCLUSIONS. EBER-1 gene expression in liver tissue precedes the occurrence of clinical and histologic PTLD. The possibility of identifying patients at risk by the method we describe here and preventing the occurrence of PTLD by a timely reduction of immunosuppression needs to be addressed by future prospective studies.     

Treatment options for post-transplant lymphoproliferative disorder and other Epstein-Barr virus-associated malignancies

Epstein-Barr virus (EBV) is associated with a range of malignancies that largely arise from a defect in EBV-specific cytotoxic T lymphocyte (CTL) immunity and function. Much work has focused on the reconstitution of CTL immunity to EBV in transplant patients, in whom immunosuppression modalities render them susceptible to post-transplant lymphoproliferative disease (PTLD). Adoptive transfer of autologous CTLs is effective at both preventing and curing PTLD in solid organ transplant recipients and can produce a long-term memory response and protection against recurring disease. In this review, the benefits and restrictions of administering EBV-specific CTLs for the treatment of PTLD are discussed and compared with emerging therapies including the generation of allogeneic human leukocyte antigen-matched CTL banks and the anti-CD20 monoclonal antibody therapy, MabThera. Furthermore, studies involving other EBV-associated disorders have described the potential benefit of adoptive transfer of EBV-specific CTLs for Hodgkin's disease, nasopharyngeal carcinoma, chronic active EBV infection, and Burkitt's lymphoma. The challenges of tailor-making therapies for individual diseases and EBV antigen expression latencies are highlighted, in addition to considering vaccination strategies for optimal treatment.     

Detection of Epstein-Barr virus DNA in sera from transplant recipients with lymphoproliferative disorders

Early diagnosis of Epstein-Barr Virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD) is important because many patients respond to reduction in immunosuppression, especially if PTLD is detected at an early stage. Previous studies have found elevated EBV DNA levels in blood from patients with PTLD, but these assays required isolation of cellular blood fractions and quantitation. We evaluated the presence of cell-free EBV DNA in serum from solid-organ transplant recipients as a marker for PTLD. Five of 6 transplant recipients with histopathologically documented PTLD had EBV DNA detected in serum at the time of diagnosis (sensitivity = 83%), compared with 0 of 16 matched transplant recipients without PTLD (specificity = 100%) (P < 0.001 [Fisher's exact test]). Furthermore, EBV DNA was detected in serum 8 and 52 months prior to the diagnosis of PTLD in two of three patients for whom stored sera were analyzed. Detection of EBV DNA in serum appears to be a useful marker for the early detection of PTLD in solid-organ transplant recipients. Further studies to define the role of such assays in evaluating solid-organ transplant patients at risk for PTLD are warranted.     

Lytic and latent EBV gene expression in transplant recipients with and without post-transplant lymphoproliferative disorder

Background

Epstein–Barr virus (EBV) is associated with post-transplant lymphoproliferative disorder (PTLD), which has significant morbidity and mortality in transplant recipients. To devise prophylactic measures, we need predictors of PTLD and a better understanding of the physiopathogenesis of the disease.

Objectives

To identify a molecular pattern of EBV gene products in blood that is specific to PTLD and can be used for the diagnosis of this disease.

Study design

We evaluated the ratio between latent and replicating EBV nucleic acids in individuals with PTLD by comparison with transplant recipients without PTLD and immunocompetent hosts with EBV DNA-emia. Subjects were prospectively identified between July 2009 and October 2010 at the University of Colorado Hospital. EBV DNA, LMP-2A Latency III and BZLF1 Lytic genes mRNA were quantified using real-time PCR.

Results

We found that PTLD subjects (N = 7) had significantly higher EBV DNA-emia compared with non-transplant immunocompetent subjects (N = 69; p < 0.0001), and transplant recipients without PTLD (N = 105; p < 0.0001). The ratios between LMP-2A and BZLF1 mRNA in transplant recipients were significantly lower than in non-transplant subjects (p = 0.04). However, PTLD and non-PTLD transplant recipients displayed similar ratios.

Conclusions

These results suggest that EBV replication makes a larger contribution to the circulating EBV DNA in transplant recipients compared with immunocompetent hosts. Transplant recipients seem to lose control over EBV replication, which may contribute to the development of PTLD.     

Presence of Epstein-Barr virus latency type III at the single cell level in post-transplantation lymphoproliferative disorders and AIDS related lymphomas.

AIMS: To investigate the expression pattern of Epstein-Barr virus (EBV) latent genes at the single cell level in post-transplantation lymphoproliferative disorders and acquired immunodefiency syndrome (AIDS) related lymphomas, in relation to cellular morphology. METHODS: Nine post-transplantation lymphoproliferative disorders and three AIDS related lymphomas were subjected to immunohistochemistry using monoclonal antibodies specific for EBV nuclear antigen 1 (EBNA1) (2H4), EBNA2 (PE2 and the new rat anti-EBNA2 monoclonal antibodies 1E6, R3, and 3E9), and LMP1 (CS1-4 and S12). Double staining was performed combining R3 or 3E9 with S12. RESULTS: R3 and 3E9 anti-EBNA2 monoclonal antibodies were more sensitive than PE2, enabling the detection of more EBNA2 positive lymphoma cells. Both in post-transplantation lymphoproliferative disorders and AIDS related lymphomas, different expression patterns were detected at the single cell level. Smaller neoplastic cells were positive for EBNA2 but negative for LMP1. Larger and more blastic neoplastic cells, sometimes resembling Reed-Sternberg cells, were LMP1 positive but EBNA2 negative (EBV latency type II). Morphologically intermediate neoplastic cells coexpressing EBNA2 and LMP1 (EBV latency type III), were detected using R3 and 3E9, and formed a considerable part of the neoplastic population in four of nine post-transplantation lymphoproliferative disorders and two of three AIDS related lymphomas. All samples contained a subpopulation of small tumour cells positive exclusively for Epstein-Barr early RNA and EBNA1. The relation between cellular morphology and EBV expression patterns in this study was less pronounced in AIDS related lymphomas than in post-transplantation lymphoproliferative disorders, because the AIDS related lymphomas were less polymorphic than the post-transplantation lymphoproliferative disorders. CONCLUSIONS: In post-transplantation lymphoproliferative disorders and AIDS related lymphomas, EBV latency type III can be detected by immunohistochemistry in a subpopulation of tumour cells using sensitive monoclonal antibodies R3 and 3E9. Our data suggest that EBV infected tumour cells in these lymphomas undergo gradual changes in the expression of EBV latent genes, and that these changes are associated with changes in cellular morphology.     

Plasma Epstein-Barr viral load predicting response after chemotherapy for post-transplant lymphoproliferative disease

Transplant patients are particularly at risk of developing B post-transplant lymphoproliferative disease (PTLD) related to intensive immunosuppressive treatment to prevent graft rejection. In EBV-positive PTLDs, EBV-DNA can be found in the patients' peripheral blood. Several methods have been described to assess peripheral blood EBV viral load. We report a case of a 13-year-old child who developed EBV-positive PTLD after renal transplantation. We assessed EBV plasma viral load by quantitative PCR and we found that the clearance of EBV-DNA correlated well with response to treatment.     

Detection of Epstein-Barr virus by polymerase chain reaction.

The polymerase chain reaction (PCR) was used to study DNA extracted from the blood of 25 transplant patients, 5 patients with infectious mononucleosis, and 13 healthy subjects and autopsy or biopsy tissue from 29 patients with lymphoproliferative disorders. Primers were directed to conserved regions of the Epstein-Barr virus (EBV) genome encoding capsid protein gp220 and Epstein-Barr nuclear antigen 1. Specific EBV amplification was found in the blood of 11 of 25 transplant patients and all patients with infectious mononucleosis. All patients with lymphoproliferative disorders occurring in the presence of immunosuppression (eight organ transplant patients and two patients with acquired immunodeficiency syndrome) had biopsies positive for EBV by PCR. Only 1 of 19 samples from lymphomas or leukemias unrelated to immunosuppression contained EBV. PCR confirmed the very close association of EBV and lymphoproliferative disorders occurring in the presence of immunosuppression. The significance of detecting EBV sequences in the blood of transplant patients, particularly in relationship to lymphoproliferation, requires further study.