Epstein-Barr virus-latent gene expression and tumor cell phenotype in acquired immunodeficiency syndrome-related non-Hodgkin's lymphoma. Correlation of lymphoma phenotype with three distinct patterns of viral latency.

We investigated 49 acquired immunodeficiency syndrome-related lymphomas (ARLs) for Epstein-Barr virus (EBV) by Southern blotting and in situ hybridization and, in positive cases, used cryostat immunohistology to compare EBV-latent gene expression (EBV encoded small RNA-1 [EBER-1], EBV nuclear antigen-2 [EBNA-2], latent membrane protein-1 [LMP-1] and host cell immunophenotype (CD11a, CD18, CD54, CD58, CD21, CD23, CD30, CD39, CDw70, immunoglobulin) patterns with those reported in other EBV infections. EBV+ immunoblast-rich/large cell ARLs (n = 22) showed three patterns of latency: broad (EBER+EBNA-2+/LMP-1+; n = 9), reminiscent of a lymphoblastoid cell line phenotype; restricted (EBER+/EBNA-2-/LMP-1-; n = 6), similar to endemic Burkitt's lymphoma; and intermediate (EBER+/EBNA-2-/LMP-1+; n = 7), a pattern rarely described in vitro but seen in certain EBV-related malignancies. EBNA-2 expression was associated with extranodal lymphomas. EBV+ Burkitt-type ARLs (n = 11) usually showed the restricted latency pattern (n = 8), but some expressed the intermediate form (n = 3). Adhesion (CD54, CD58) and activation (CD30, CD39, CDw70) molecule expression varied with morphology (immunoblast-rich/large cell versus Burkitt-type), but was not independently correlated with EBV-positivity. CD30 and LMP-1 expression were associated. ARLs show heterogeneity regarding both the presence of EBV and latency pattern. Comparison of these phenotypically distinct lymphoma groups with known forms of EBV infection provides clues to their possible pathogenesis.     

Down-regulation of Epstein-Barr virus nuclear antigen 1 in Reed-Sternberg cells of Hodgkin's disease.

AIMS--To demonstrate Epstein-Barr virus (EBV) encoded nuclear antigen 1 (EBNA-1) gene expression in EBV associated disorders using a new monoclonal antibody (1H4-1) on routinely processed tissues. METHODS--The pressure cooker antigen retrieval method was used for the immunohistochemical demonstration of EBNA-1 gene expression in formalin fixed, EBV positive tissues from Hodgkin's disease, infectious mononucleosis, HIV associated non-Hodgkin's lymphomas, post-transplant lymphomas, and undifferentiated nasopharyngeal carcinoma (NPC). EBV encoded EBNA-2, latent membrane protein 1 (LMP-1) and BZLF-1 gene expression was also examined using commercially available monoclonal antibodies. RESULTS--Of the 34 EBER in situ hybridisation positive cases of Hodgkin's disease examined, none expressed EBNA-1 in the Reed-Sternberg cells. These cells were nevertheless strongly LMP-1 positive in all cases. Strong EBNA-1 staining was seen in all cases of EBER positive HIV associated non-Hodgkin's lymphoma (five of five), nasopharyngeal carcinoma (five of five), infectious mononucleosis (three of three), and post-transplant lymphoma (one of one). These cases also expressed LMP-1, EBNA-2 and BZLF-1, but at differing levels. CONCLUSION--The pressure cooker antigen retrieval procedure is a sensitive and reliable adjunct to immunohistochemistry, especially with antibodies which are otherwise ineffective on routinely processed tissues. The EBNA-1 gene is not expressed at detectable levels in the malignant cells of Hodgkin's disease, but is consistently expressed in other EBV associated disorders. This finding has important implications for the role of EBNA-1 in the biology of EBV.     

Enterocytozoon bieneusi as a cause of proliferative serositis in simian immunodeficiency virus-infected immunodeficient macaques (Macaca mulatta)

CONTEXT: Enterocytozoon bieneusi is the most frequent microsporidian parasite of human patients with acquired immunodeficiency syndrome and is a significant cause of diarrhea and wasting. Recently, this organism has also been recognized as a spontaneous infection of several species of captive macaques. As in humans, E bieneusi frequently causes enteropathy and cholangiohepatitis in immunodeficient simian immunodeficiency virus (SIV)-infected macaques. OBJECTIVE: To examine E bieneusi as an etiologic agent of nonsuppurative proliferative serositis in immunodeficient rhesus macaques (Macaca mulatta). DESIGN: Retrospective analysis of necropsy material obtained from immunodeficient SIV-infected rhesus macaques. RESULTS: Examination of SIV-infected rhesus macaques (n = 225) revealed E bieneusi proliferative serositis in 7 of 16 cases of peritonitis of unknown origin. The organism could be identified by in situ hybridization and polymerase chain reaction in sections of pleura and peritoneum obtained at necropsy. Serositis was always accompanied by moderate-to-severe infection of the alimentary tract, and morphologic evidence suggested dissemination through efferent lymphatics. Colabeling experiments revealed most infected cells to be cytokeratin positive and less frequently positive for the macrophage marker CD68. Sequencing of a 607-base pair segment of the small subunit ribosomal gene revealed 100% identity to sequences obtained from rhesus macaques (Genbank accession AF023245) and human patients (Genbank accession AF024657 and L16868). CONCLUSIONS: These findings indicate that E bieneusi disseminates in immunodeficient macaques and may be a cause of peritonitis in the immunocompromised host.     

Epstein-Barr virus in B-cell non-Hodgkin's lymphomas: Unexpected infection patterns and different infection incidence in low- and high-grade types

Two hundred and eight cases of B-cell non-Hodgkin's lymphoma (B-NHL) occurring in Europeans without any signs of HIV infection were investigated for their association with an Epstein-Barr virus (EBV) infection. The polymerase chain reaction (PCR) was applied for EBV-DNA detection, in situ hybridization (ISH) for the cellular localization of EBV-encoded small nuclear RNAs (EBER) and immediate-early RNAs (BHLF), and immunohistology (IH) for the detection of EBV-encoded latent membrane protein (LMP) and EBV nuclear antigen 2 (EBNA2) expression. PCR and EBER-ISH produced congruent results in those cases with amplifiable DNA. EBV was present overall in 26 per cent (54/208) of the B-NHL cases. Through EBER-ISH, the virus could be localized merely in rare non-neoplastic bystander lymphocytes in 27 and additionally in tumour cells of 27 cases. Unexpectedly, the proportion of EBV-infected tumour cells present in the different cases varied between 1 and 100 per cent. All but three of the cases with infected tumour cells were of high-grade malignancy. Correlation with the morphological and immunological tumour phenotype revealed that all cases with more than 80 per cent EBER-positive tumour cells were either B-anaplastic large cell lymphomas (B-ALCL), sporadic Burkitt's lymphomas, or B-NHLs with partial or full plasmacellular differentiation. LMP was consistently absent from Burkitt's lymphomas and constantly expressed in B-ALCLs with EBER-positive tumour cells, while in all other instances it varied greatly and was rarer than EBER expression. The above findings suggest that in cases where EBV is present in 80–100 per cent of tumour cells, EBV infection takes place before malignant transformation and thus may have contributed to the malignant phenotype, whereas in the other cases with only a smaller number of infected tumour and single bystander cells, EBV infection may have occurred following malignant transformation. In these cases, infection would appear to be of little or no significance in tumourigenesis.     

Epstein-Barr virus in nasal T-cell lymphomas (polymorphic reticulosis/midline malignant reticulosis) in Western China

Polymorphic reticulosis (PR) or midline malignant reticulosis (MMR) is considered to be malignant, or at least pre-malignant T-cell proliferations of the nose or midline area. Recent reports of small series of nasal T-cell lymphomas have shown a strong association with Epstein-Barr virus (EBV). Furthermore, a peculiar phenotype is described, with expression of CD56 and not of CD3, suggesting a possible origin from natural killer (NK) cells. We have analysed a series of 38 cases of PR/MMR for the presence of EBV by in situ hybridization (ISH) of the EBV-encoded RNAs 1 and 2 (EBER). Twenty cases were tested for expression of EBV-encoded latent membrane protein 1 (LMP-1). Special attention was also paid to the expression of CD3 and the NK cell-related marker CD56. Thirty-two cases (84 per cent) showed positive EBER ISH. In 5 of 20 cases, LMP-1 expression was detected. In three cases, a few scattered cells were positive, and in two cases, LMP-1 was detected in clusters of atypical cell. Most of the neoplasms showed expression of CD3 (89 per cent) and in 27 cases (71 per cent), CD56 was detected. These results are consistent with an aetiopathogenetic role for EBV in most, but not all, cases of PR/MMR. Our findings are less supportive of a major role for LMP-1 in tumour genesis. CD3 expression in most of the cases of PR/MMR underlines the T-cell origin of these neoplasms, often with aberrant expression of CD56.     

Cold agglutinins in rhesus macaques infected with simian immunodeficiency virus (SIV)

Cold agglutinins are uncommon autoimmune phenomena associated with mycoplasma pneumonia, viral infections, lymphoproliferative neoplasia and recently, acquired immunodeficiency syndrome (AIDS) in human beings. Six rhesus macaques infected with pathogenic isolates of simian immunodeficiency virus (SIV) developed cold agglutinins late in the course of viral infection in association with hyperproteinaemia, hyperglobulinaemia and thrombocytopenia. Cold agglutinin titres ranged from 1:1024 to 1:8192. The development of cold agglutinins in SIV-infected rhesus macaques may be another manifestation of immune dysfunction in this non-human primate model of AIDS.     

Frequent expansion of Epstein–Barr virus (EBV) infected cells in germinal centres of tonsils from an area with a high incidence of EBV-associated lymphoma

Burkitt's lymphoma (BL) and Hodgkin's disease (HD) occurring in developing regions are frequently associated with Epstein–Barr virus (EBV) infection and have a high incidence in childhood. Recent genotyping studies indicate that the tumour cells of both neoplasms represent B cells that contain somatically mutated immunoglobulin heavy chain genes. This implies that the precursors of these neoplasms have participated in the germinal centre (GC) reaction. We therefore presumed that normal lymphoid tissues from children living in developing regions would harbour an increased number of EBV-infected cells within the GC, when compared with children living in industrialized nations. To test this hypothesis, hyperplastic tonsils from 40 children living in Bahia (Brazil) and 40 from German children were analysed for the presence of EBV-encoded small nuclear RNA (EBER) and EBV-encoded proteins by in situ hybridization and immunohistology, respectively. Although the overall EBV infection rate was similar in both groups (50 per cent of Bahian vs. 45 per cent of German cases), a significantly higher number of EBER-positive lymphoid cells were found in the GCs of 8/20 EBV-positive tonsils from Brazil (9–89 cells/GC; mean: 14·5 cells/GC per case), while only 3/18 tonsils from Germany displayed a few EBER positive cells (1–9 cells/GC; mean: 0·5 cell/GC per case) in this compartment (p < 0·007). In addition, the EBV-infected GC cells in Bahian samples resembled centroblasts, exhibited mitotic activity, and in two cases showed expression of EBV-encoded latent membrane protein (LMP)-1, findings not present in German samples. These data show that latently EBV-infected cells participate more frequently in GC reactions in developing regions than in industrialized countries and may abnormally express the oncogenic protein LMP-1. This could in part explain the higher incidence in this region of EBV association with lymphomas related to GC cells or their progeny, such as BL and HD. Copyright © 1999 John Wiley & Sons, Ltd.     

No significant association of Epstein-Barr virus infection with invasive breast carcinoma

We studied 48 cases of invasive breast carcinoma for evidence of Epstein-Barr virus (EBV), which is associated with many human malignancies. In situ hybridization studies to detect the presence of EBV-encoded small nonpolyadenylated RNA (EBER)-1 were performed in paraffin sections. Immunohistochemical studies to detect EBV nuclear antigen (EBNA)-1, latent membrane protein (LMP)-1, and the transactivating immediate-early BZLF1 (ZEBRA) protein were also performed in paraffin sections. The presence of EBV genomic DNA was studied by polymerase chain reaction (PCR) amplification using sets of primers flanking the EBNA-4 and the EBV-LMP-1 genes in frozen tissues. Southern blot analysis using a probe flanking the EBV terminal repeat region was then attempted in cases that were PCR-positive. Five of 48 cases (10%) of breast carcinoma showed focal EBER-positive tumor cells. Twelve cases (25%) were positive for EBNA-1 by immunohistochemistry, all but one different from the EBER-positive cases. None of the cases were positive for LMP-1 or ZEBRA protein by immunohistochemistry. PCR studies for EBNA-4 and LMP-1 were each positive in five cases (including three cases in common). However, Southern blot studies successfully performed in all but one of the PCR-positive cases were completely negative. The identification of EBV by any methodology was not correlated with tumor size, grade, or lymph node status. This study demonstrated evidence of EBV infection in tissues involved by invasive breast carcinomas in a significant subset of cases. However, the lack of localization of EBV infection to a significant population of the tumor cells in any case, the negativity by Southern blot hybridization, and the lack of expression of multiple antigens in any case strongly argue against a significant role for EBV in the pathogenesis of breast carcinoma.     

Pathogenesis of SIV encephalitis. Selection and replication of neurovirulent SIV.

To investigate the viral and host factors that contribute to neurological disease, nine macaques were intravenously co-inoculated with SIV/DeltaB670, a primary isolate of SIV consisting of at least 21 different genotypes, and SIV/17E-Fr, a neurovirulent recombinant clone. CD4+ cell counts and antigenemia were measured throughout infection. The SIV env V1 region was amplified from brain and peripheral blood mononuclear cell DNA to compare the genotypes present in brain and blood. Seven of the 9 macaques (78%) developed typical SIV-associated neurological lesions classified as severe (4 macaques), moderate (2 macaques), or mild (1 macaque) with a mean time to euthanasia of 7 months. Macaques with severe neurological lesions progressed more rapidly, with a mean time to euthanasia of 3-6 months. SIV/17E-Fr was detected in brain homogenates from all four macaques with severe encephalitis, and in three of the four, SIV/17E-Fr was the only genotype identified in the central nervous system. Macaques with less severe or no neurological lesions usually had one of various genotypes of SIV/DeltaB670 in brain. A variety of genotypes of SIV/DeltaB670 and SIV/17E-Fr were detected in peripheral blood mononuclear cells throughout infection. Macaques with severe neurological lesions had the most precipitous declines in CD4+ cell counts, the highest levels of antigenemia, and the greatest expression of viral RNA and protein in the central nervous system. Macaca nemestrina were more likely to develop severe neurological lesions than M. mulatta or M. fascicularis (P = 0.048). This study demonstrated that neurovirulent strains within the virus swarm can selectively enter and become established in the central nervous system and that the neurological lesions that develop are correlated with the development of host immunosuppression. The species differences in severity of neurological lesions seen in this study suggest that host factors are also important in determining the outcome of lentiviral infection.     

CD21-independent Infection of Epstein-Barr Virus in Human Signet Ring Gastric Carcinoma Cell Line

Epstein Barr virus ( EBV), a ubiquitous human her pesvirus, is the etiologic agent of infectious mononucleo sis, and is closely associated with several human malig nancies including Burkitt′s lymphoma (BL), undifferenti ated nasopharyngeal carcinoma (NPC) and opportunisticlymphoma in immunocompromised hosts. In recent years,there have been increasing evidences of association of EBVwith additional malignancies, such as gastric carcinoma.EBV has been found in tumor cells of m…     

Characteristics of Epstein-Barr virus isolated from the malignant lymphomas in Korea

Epstein-Barr virus (EBV) is a common herpes virus linked to a variety of human neoplasms. In this study, the EBV detection was identified with the paraffin-embedded tissues from 62 non-Hodgkin's lymphomas, 20 Hodgkin's lymphomas, and 48 non-neoplastic tonsils, using PCR for EBNA-1 and EBER-1 mRNA in situ hybridization for EBER-1 mRNA. The isolates were analyzed for type 1/2, variants C/D and F/f, and LMP-1 30 bp deletion. EBV was isolated in 31 of 48 (66%) non-neoplastic tonsils, 24 of 42 (57%) B cell lymphomas, in 15 of 20 (75%) T cell lymphomas, and 17 of 20 (85%) Hodgkin's lymphomas. These viruses were classified as type 1 for 81% of non-neoplastic tonsils, 95% of B cell lymphomas, 93% of T cell lymphomas, and 73% of Hodgkin's diseases. Both type 1 and 2 viruses were isolated in one non-neoplastic tonsil and 3 Hodgkin's diseases. Type C virus was predominant in non-neoplastic tonsils (77%) and B cell lymphomas (75%), while type D virus was common in T cell lymphomas (71%) and Hodgkin's diseases (73%) (P < 0.05). Majority of the viruses detected in non-neoplastic tonsils (93%) and malignant lymphomas (91%) were "F" prototype. LMP-1 30 bp deletion was found in high frequency in both non-neoplastic tonsils (92%) and malignant lymphomas (86%). In conclusion, most of EBV found in Korea was type 1, and "DF" genotype was more frequent in T cell lymphomas and Hodgkin's diseases than in non-neoplastic tonsils and B cell lymphomas. LMP-1 30 bp deletion did not seem to be associated with malignant lymphomas.     

Characteristics of Epstein-Barr virus associated childhood non-Hodgkin’s lymphoma in the Republic of Korea

To analyze the clinicopathologic characteristics of childhood non-Hodgkins lymphoma (NHL) associated with Epstein-Barr virus (EBV), EBER in situ hybridization was performed in 80 cases of NHLs. EBER-positive lymphomas account for 25% (20/80) and include NK/T-cell lymphoma (6/6), aggressive NK-cell leukemia (1/1), peripheral T cell lymphoma (5/11), diffuse large B-cell lymphoma (5/14), hydroa-like T-cell lymphoma (1/1), marginal zone B-cell lymphoma (1/2), and post-transplantation lymphoproliferative disorder (1/1). Other types including 19 cases of Burkitts lymphoma were negative. For 9 EBER-positive cases, immunohistochemical staining for LMP-1, and EBNA-2 was performed to determine the EBV latency pattern. Two of nine EBER-positive cases expressed both LMP-1 and EBNA-2. Clinically, patients with EBV-positive B-cell lymphomas were cured with chemotherapy, whereas EBV-associated NK- and T cell lymphomas pursued fatal clinical course. In conclusion, EBVs infected in childhood NHLs are frequently associated not only with NK- and T- cell lymphomas but also large B-cell lymphomas.     

Longitudinal analysis of monocyte/macrophage infection in simian immunodeficiency virus-infected, CD8+ T-cell-depleted macaques that develop lentiviral encephalitis

The histopathological hallmark of lentiviral-associated encephalitis is an abundance of infected and activated macrophages. Why a subset of infected hosts develops lentiviral encephalitis and others do not is unknown. Using a CD8(+) T-cell depletion model of simian immunodeficiency virus (SIV)-infected rhesus macaques, we examined the relationship between peripheral SIV infection of monocytes/macrophages and the development of encephalitis. At the same time that cerebral spinal fluid viral load increased in macaques that developed encephalitis, we observed that monocyte-derived macrophages from these macaques produced more virus than those from macaques that did not develop encephalitis. However, during the course of infection, the number of blood monocyte-associated SIV DNA copies did not distinguish macaques that developed simian immunodeficiency virus encephalitis from macaques that did not develop encephalitis. Paradoxically, in this model, macaques that developed encephalitis had fewer SIV-infected macrophages in lungs and thymus at postmortem than macaques that did not develop encephalitis. These findings suggest that inherent differences in host monocyte viral production are related to development of encephalitis.     

Inconsistent association of Epstein-Barr virus with CD56 (NCAM)-positive angiocentric lymphoma occuring in sites other than the upper and lower respiratory tract

We previously described nine cases of angiocentric lymphoma of a possible natural killer (NK)-cell lineage with a surface CD3− CD56+ phenotype occurring in sites other than the upper and lower respiratory tract. This study was performed to investigate the association of Epstein-Barr virus (EBV) with these lymphomas, using the polymerase chain reaction (PCR) for the presence of EBV-DNA, in situ hybridization (ISH) for EBV-encoded small RNAs (EBERs) and immunohistology for EBV-determined nuclear antigen-2 (EBNA-2) and latent membrane protein-1 (LMP-1) in paraffin sections. PCR and ISH produced almost identical results, and EBERs were identified in the nuclei of the lymphoma cells of three cases, two of which exhibited LMP-1 in the cytoplasm of tumour cells without EBNA-2 expression. Molecular genetic analysis revealed EBV to be incorporated into these three EBER-positive cases either clonally or biclonally. It was revealed by re-evaluation of their morphology with the established EBV status on each case that, in contrast to the rather variable and irregular cellular composition of the EBV- positive tumours, the EBV-negative tumours stood out because of their remarkably uniform 'blastoid' appearance, and could be grouped as blastic NK-cell lymphoma. The relationship of the EBV-positive cases with nasal NK-cell tumours has yet to be clarified.     

Increased number of Epstein-Barr virus latently infected B-cells in T-cell non-Hodgkin's lymphoma tissues

Summary Epstein-Barr virus (EBV) is a causative agent of malignant lymphomas occurring in immunocompromised hosts. Similar lymphoid tumors can be induced in mice with severe combined immunodeficiency (SCID mice) by transplanting human B-cells with latently infected EBV. We have previously observed that when apparently EBV-negative lymphomas were engrafted into SCID mice, 11 of 18 T-cell non-Hodgkin's lymphomas (NHLs) produced EBV associated lymphomas, but only 2 of 30 engrafted with B-NHLs. Previous studies suggested that EBV-infected cell inducing lymphomas in SCID mice may preferentially exist in T-cell NHL tissues. To prove this assumption, in situ hybridization (ISH) using oligonucleotide probes for EBV-encoded small RNAs 1 (EBER1) was used in this study to demonstrate EBV-bearing lymphocytes in NHL tissues. It was found that EBV-bearing cells existe in 9 of the 10 T-cell NHL surgical specimens. By contrast, in B cell NHLs, only 2 of 10 carried EBV-bearing cells. Further semi-quantitative analysis demonstrated that apparently significantly more EBV-bearing cells were present in T-cell NHL tissues than in B-cell NHLs. Moreover, these EBV-bearing cells in lymphoma tissues were shown to be of B-cell lineage, by the combinated analysis of immunostaining with CD20 and ISH with EBER1. These results indicated the increase of EBV-bearing B-cells in T-cell NHL tissues, suggesting the activation of B-cells with latently infected EBV by neoplastic T-cells.     

Survival and failure to thrive in the SIV-infected juvenile rhesus monkey

In AIDS patients, wasting in adults and failure to thrive in children are common and devastating problems. Weight loss in rhesus macaques infected with simian immunodeficiency virus (SIV) has not been well characterized. The purpose of this study was to determine growth curves in SIV-infected juvenile macaques to determine the effects of SIV infection on body weight and growth. Medical records of seven juvenile male SIV-infected macaques were retrospectively reviewed to determine body weights, survival time, CD4 count, and viral load. Mean age and body weight at the time of inoculation were 63.3 weeks and 2.4 kg, respectively. Mean survival was 73.7 weeks, and mean body weight at the time of death was 3.0 kg, whereas the published mean body weight for this age of male rhesus macaque is 4.1 kg. Compared with the linear growth pattern of normal animals, the growth pattern for the SIV-infected animals exhibited strong nonlinearity with an inflection point at the mean survival of 74 weeks. After this time point, the discrepancy between growth curves for infected and healthy animals increased at a greater rate. Body weight correlated inversely with viral load (r = -0.368; p = .003) but there was no correlation between body weight and CD4 count. The results of this study suggest that failure to thrive is a consequence of SIV infection and may be related to severity of infection.     

Early events in Epstein-Barr virus infection of human B lymphocytes.

The sequence of Epstein-Barr virus (EBV) and B lymphocyte changes in the 3 days following acute infection was analyzed. By 16 hr the average infected lymphocyte had 1 EBV episome. Nuclear protein-2 (EBNA-2) and EBNA-leader protein (-LP) were detected by 12 hr, and by 32 hr were at the levels of stable EBV infection in lymphoblastoid cell lines (LCLs). At 12 hr, all EBNA-LP and EBNA-2 RNAs were initiated from the Pw promoter. By 36 hr a significant EBNA-LP and EBNA-2 RNA fraction initiated from the upstream Pc promoter. Throughout acute infection, a similar fraction of potentially bicistronic EBNA-LP mRNAs had first exon splices which would result in EBNA-LP translation. By 36 hr c-myc RNA was transiently induced, and CD21 and CD23 RNAs were beginning to increase. This coincided with low-level EBNA-1, EBNA-3A, B, and C, and latent membrane protein-1 (LMP-1) expression. By 46 hr, EBNA-1, the EBNA-3s, and LMP-1 were near the levels ordinarily found in LCLs and a substantial fraction of lymphocytes were in S phase. These results are compatible with a key role for EBNA-2 (or EBNA-LP) in regulating virus and cell gene expression. High-level expression of the EBV-encoded small RNAs, EBERs, was delayed beyond 36 hr and may, therefore, be activated by other virus or cell genes. A 65-kDa virion protein persisted in acutely infected cells. This protein could be a mediator of virus or cell gene expression.