Epstein-Barr Virus Genomic DNA Research Articles

Decitabine disrupts EBV genomic epiallele DNA methylation patterns around CTCF binding sites to increase chromatin accessibility and lytic transcription in gastric cancer.

Epstein-Barr virus (EBV) latency is controlled by epigenetic silencing by DNA methylation [5-methyl cytosine (5mC)], histone modifications, and chromatin looping. However, how they dictate the transcriptional program in EBV-associated gastric cancers remains incompletely understood. EBV-associated gastric cancer displays a 5mC hypermethylated phenotype. A potential treatment for this cancer subtype is the DNA hypomethylating agent, which induces EBV lytic reactivation and targets hypermethylation of the cellular DNA. In this study, we identified a heterogeneous pool of EBV epialleles within two tumor-derived gastric cancer cell lines that are disrupted with a hypomethylating agent. Stochastic DNA methylation patterning at critical regulatory regions may be an underlying mechanism for spontaneous reactivation. Our results highlight the critical role of epigenetic modulation on EBV latency and life cycle, which is maintained through the interaction between 5mC and the host protein CCCTC-binding factor.

Methionine metabolism controls the B cell EBV epigenome and viral latency

Epstein-Barr virus (EBV) subverts host epigenetic pathways to switch between viral latency programs, colonize the B cell compartment, and reactivate. Within memory B cells, the reservoir for lifelong infection, EBV genomic DNA and histone methylation marks restrict gene expression. But this epigenetic strategy also enables EBV-infected tumors, including Burkitt lymphomas, to evade immune detection. Little is known about host cell metabolic pathways that support EBV epigenome landscapes. We therefore used amino acid restriction, metabolomic, and CRISPR approaches to identify that an abundant methionine supply and interconnecting methionine and folate cycles maintain Burkitt EBV gene silencing. Methionine restriction, or methionine cycle perturbation, hypomethylated EBV genomes and de-repressed latent membrane protein and lytic gene expression. Methionine metabolism also shaped EBV latency gene regulation required for B cell immortalization. Dietary methionine restriction altered murine Burkitt xenograft metabolomes and de-repressed EBV immunogens invivo. These results highlight epigenetic/immunometabolism crosstalk supporting the EBV B cell life cycle and suggest therapeutic approaches.

Association of Epstein-Barr virus latently expressed genes with multiple sclerosis

BackgroundDespite mounting evidence supporting an etiologic role for Epstein-Barr virus (EBV) in multiple sclerosis (MS), the exact mechanisms through which the virus may contribute to disease development are still unknown. The aim of this study was to analyze seven highly polymorphic EBV latently expressed genes in individuals diagnosed with MS in comparison to healthy controls (HC), to investigate the possible association of EBV variants with an individual's risk towards MS. MethodsB-lymphocytes were isolated from MS patients (n = 30) and HC (n = 33) for the isolation of EBV genomic DNA. Sanger sequencing was employed to analyze EBV latent gene regions. ResultsA total of 26 variants were detected in our cohort, 17 of which were significantly associated with the MS group while nine were significantly associated with HC. Following the designation of EBV alleles based on these variants, MS risk was found to be significantly associated with the presence of the EBNA3B2.1 allele (p = 0.0008) and LMP1.1 allele (p = 0.01), whereas the EBNA1.3 allele (p = 0.005), EBNA2.1 allele (p = 0.001) as well as the EBNA3B2.2 allele (p = 0.0003) appeared to provide a protective role. ConclusionsThis study indicates a marked association between EBV genetic variants and MS, lending further support towards possible molecular mechanisms through which EBV may contribute to disease development.

Application of Biopsy Samples Used for Helicobacter pylori Urease Test to Predict Epstein-Barr Virus-Associated Cancer.

Persistent gastric mucosal damage caused by Helicobacter pylori infection is a major risk factor for gastric cancer (GC). The Epstein–Barr virus (EBV) is also associated with GC. Most patients with EBV-associated GC are infected with H. pylori in East Asia. However, very few reports have described where and when both H. pylori and EBV infect the gastric mucosa. To clarify this, old biopsy samples used for the rapid urease test (RUT) were applied to count EBV genomic DNA (gDNA) copies using DNA probe quantitative polymerase chain reaction. DNA extracted from the gastric biopsy samples of 58 patients with atrophic gastritis was used to analyze the correlation between the degree of atrophic gastritis and the copy number of EBV gDNA. EBV was detected in 44 cases (75.9%), with viral copy numbers ranging from 12.6 to 4754.6. A significant correlation was found between patients with more than 900 copies of EBV gDNA and those with a more severe grade of atrophic gastritis (p = 0.041). This study shows that EBV can be detected in RUT samples in a manner that reduces patient burden.

The Hsp70 inhibitor 2-phenylethynesulfonamide inhibits replication and carcinogenicity of Epstein\u2013Barr virus by inhibiting the molecular chaperone function of Hsp70

Epstein–Barr virus (EBV) can infect cells in latent and lytic period and cause serious disease. Epstein–Barr virus nuclear antigen 1 (EBNA1) is essential for the maintenance of the EBV DNA episome, replication and transcription. 2-phenylethynesulfonamide (PES) is a small molecular inhibitor of Heat shock protein 70 (Hsp70), which can interact with Hsp70 and disrupts its association with co-chaperones and substrate proteins of Hsp70. In our study, we found that PES could decrease the expression of EBNA1, which is independent of effects on EBNA1 transcription or proteasomal degradation pathway. The central glycine–alanine repeats domain was not required for inhibition of EBNA1 expression by PES. Also, PES could reduce the amount of intracellular EBV genomic DNA. PES inhibited proliferation and migration but induced cell cycle arrest and apoptosis of EBV positive cells. In addition, silencing of Hsp70 decreased expression of EBNA1 and the amounts of intracellular EBV genomic DNA, and PES increased this effect on a dose-dependent manner. On the contrast, over-expression of Hsp70 enhanced the expression of EBNA1 and the amounts of intracellular EBV genomic DNA, but PES inhibited this effect on a dose-dependent manner. Furthermore, Hsp70 interacted with EBNA1 but PES interfered this interaction. Our results indicate that PES suppresses replication and carcinogenicity of Epstein–Barr virus via inhibiting the molecular chaperone function of Hsp70.

Quantitative PCR for Plasma Epstein-Barr Virus Loads in Cancer Diagnostics.

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is associated with posttransplant lymphoproliferative disease (PTLD), Hodgkin's lymphoma, Burkitt's lymphoma, nasopharyngeal carcinoma, and HIV-related lymphomas. It infects nearly all humans and then persists for the life of the host in a small proportion of benign B lymphocytes. EBV reactivation, usually in the setting of immunosuppression, is the main risk factor for development of EBV-associated malignancies. EBV reactivation can be detected in tissue specimens using EBV-encoded RNA (EBER) in situ hybridization (ISH), which is routinely used for diagnosis of PTLD and nasopharyngeal carcinoma. However, EBER ISH cannot be routinely used for screening asymptomatic or monitoring posttreatment outcome due to difficulty in obtaining tissue specimens for testing and the nonquantitative nature of the assay. Recent studies have shown that EBV genomic DNA can be detected in blood of patients with EBV-associated diseases, and that monitoring of EBV viral load in blood could be an effective method of distinguishing disease-associated EBV reactivation from incidental EBV present in benign B lymphocytes, and could be used for diagnostic screening and monitoring of EBV-associated diseases. In this chapter we discuss a protocol for quantitative plasma EBV DNA analysis.

New Insight into Epstein-Barr Virus-Associated Biological Behavior of Aggressive Natural Killer Cell Leukemia

Aggressive natural killer cell leukemia (ANKL) is a rare and highly aggressive subtype of mature NK-cell neoplasms. Similar with extranodal NK/T-cell lymphoma, nasal type (ENKL), another subtype of NK-cell neoplasm, ANKL is also an Asian-prevalent and Epstein-Barr virus (EBV)-related neoplasm. In contrast, our knowledge of ANKL, especially about EBV biological behavior in this rare leukemia, lags far behind that of ENKL and other EBV-related hematopoietic malignancies, such as Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and post-transplant lymphoproliferative disorder (PTLD). Dissection of the virus-host crosstalk in ANKL could contribute to better understanding the mechanism and finding out effective therapy for this neoplasm.In the present study, we investigated EBV-associated biological behavior in serial ANKL patients, including the clinical presentation, EBV genomic DNA, EBV antigens expression, cytogenetic-molecular aberrations, and leukemia-associated microenvironment. A total of 28 ANKL patients were collected upon review of the clinical database in Nanfang hospital. Different items of EBV infection evidence consisted of EBV viremia (n=9), EBV genomic DNA (n=20), and EBER/EBNA/LMP1/LMP2A expression (n=23). EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) was the predominant clinical feature. Bone marrow smear was infiltrated with large granular lymphocyte (LGL) with LMP1/LMP2a-positive bulb, indicating the presence of EBV viral inclusions bodies. Positron emission tomographic (PET)-computed tomographic (CT) scan revealed bone marrow, liver and spleen as the most frequently involved organs, compared with nose and nasopharynx in ENKL. Cytogenetic analysis demonstrated 7q10-32 (n=4) was the “hotspot” of chromosome aberrations in ANKL. PCR analysis with EBNA-2/LMP1 specific primers on reserved DNA samples (n=20) revealed ANKL cells were infected with type-1 EBV strain with wide-type LMP1 (n=20), compared with 30bp-deleted LMP1 gene in ENKL. Integrated mutation analysis (n=20) identified recurrent mutations in Src homology 2 (SH2) domains of STAT5a (n=7) and p16inka (exon 3/4, n=20), but no mutation in SH2 domains of ID2, STAT1, and STAT3. Immunochemical (IHC) analysis on formalin-fixed paraffin-embedded tissues (n=23) revealed latency type-3 EBV expression in ANKL cells, with latency antigens of EBER, EBNA, LMP-1, and LMP-2. Furthermore, LMP-1/LMP-2-positive leukemia/lymphoma-associated macrophages (LAMs, n=23) were enriched in ANKL microenvironment. Notably, EBV-positive LAMs were significantly associated with poor prognosis and disease progression. Univariate analysis revealed significant difference (p<0.05) in overall survival (OS) between High-Ratio of CD68+LAMs/CD56+ANKLs (HMA, n=13) and Low-Ratio LAM/ANKLs cohorts (LMA, n=10). Furthermore, IHC analysis on paired presentation and progression samples (n=3) showed that EBV-positive LAMs increased in pace with disease progression.Conclusions: Our data demonstrate that type-1 EBV strain as being latency type-3 expression infected both leukemia cells and microenvironment and thus linked the pathogen-microenvironment-host crosstalk in ANKL. EBV-infected leukemia-associated microenvironment, particularly LAMs, might not only play a critical role in prognosis classification but also contribute to the leukemogenesis of NK transformation in ANKL, which it’s still poorly understood and deserves more research efforts. DisclosuresZhou:Guangzhou Pearl River of Science and Technology New Star Project: Research Funding.

Tumour inflammasome‐derived IL‐1β recruits neutrophils and improves local recurrence‐free survival in EBV‐induced nasopharyngeal carcinoma

Inflammasomes sense infection and cellular damage and are critical for triggering inflammation through IL-1β production. In carcinogenesis, inflammasomes may have contradictory roles through facilitating antitumour immunity and inducing oncogenic factors. Their function in cancer remains poorly characterized. Here we show that the NLRP3, AIM2 and RIG-I inflammasomes are overexpressed in Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), and expression levels correlate with patient survival. In tumour cells, AIM2 and RIG-I are required for IL-1β induction by EBV genomic DNA and EBV-encoded small RNAs, respectively, while NLRP3 responds to extracellular ATP and reactive oxygen species. Irradiation and chemotherapy can further activate AIM2 and NLRP3, respectively. In mice, tumour-derived IL-1β inhibits tumour growth and enhances survival through host responses. Mechanistically, IL-1β-mediated anti-tumour effects depend on infiltrated immunostimulatory neutrophils. We show further that presence of tumour-associated neutrophils is significantly associated with better survival in NPC patients. Thus, tumour inflammasomes play a key role in tumour control by recruiting neutrophils, and their expression levels are favourable prognostic markers and promising therapeutic targets in patients.

Characterization of Epstein Barr Virus Latency Pattern in Argentine Breast Carcinoma

IntroductionEpstein-Barr virus (EBV)-associated tumors show different expression patterns of latency genes. Since in breast carcinoma this pattern is not yet fully described, our aim was to characterize EBV latency pattern in our EBV positive breast carcinoma series.MethodsThe study was conducted on 71 biopsies of breast carcinoma and in 48 non-neoplastic breast controls. EBNA1, LMP2A and LMP1 expression was assessed by immunohistochemistry with monoclonal antibodies, while viral genomic DNA and EBERs RNA transcripts expression was performed by in situ hybridization. EBV presence was confirmed by PCR.ResultsEBV genomic DNA and EBNA1 expression were detected in 31% (22/71) of patients specifically restricted to tumor epithelial cells in breast carcinoma while all breast control samples were negative for both viral DNA and EBNA1 protein. LMP2A was detected in 73% of EBNA1 positive cases, none of which expressed either LMP1 protein or EBERs transcripts.ConclusionsThese findings suggest that EBV expression pattern in the studied biopsies could be different from those previously observed in breast carcinoma cell lines and lead us to suggest a new, EBNA1, LMP2A positive and LMP1 and EBERs negative latency profile in breast carcinoma in our population.

Validation of Roche LightCycler Epstein-Barr Virus Quantification Reagents in a Clinical Laboratory Setting

Epstein-Barr virus (EBV) is associated with a wide range of benign and malignant diseases, including infectious mononucleosis, lymphoma, posttransplant lymphoproliferative disorder, and nasopharyngeal carcinoma. Measurement of EBV viral load in plasma is increasingly used for rapid assessment of disease status. We evaluated the performance characteristics of an EBV polymerase chain reaction assay that uses commercial reagents and instruments from Roche Diagnostics (Indianapolis, IN). DNA was extracted from plasma using a MagNaPure instrument, and viral load was measured by real-time polymerase chain reaction on a LightCycler. Analyte-specific reagents included primers and hybridization probes targeting the EBV LMP2 gene and a spiked control sequence. Accuracy and reproducibility were established using DNA from three cell lines. The assay was sensitive to approximately 750 copies of EBV DNA per milliliter of plasma and was linear across at least four orders of magnitude. The assay detected EBV DNA in three of five samples from nasopharyngeal carcinoma patients, seven of nine infectious mononucleosis samples, and 34/34 samples from immunosuppressed patients with clinically significant EBV-related disease, whereas EBV DNA was undetectable in plasma from 21 individuals without EBV-related disease. In conclusion, this LightCycler EBV assay is rapid, sensitive, and linear for quantifying EBV viral load. The assay appears to be useful for measuring clinically significant EBV levels in immunodeficient patients.

Latent and lytic Epstein-Barr virus replication strategies.

The Epstein-Barr virus (EBV) can choose between two alternative lifestyles; latent or lytic replication. In the latent state, the EBV genomic DNA, which exists as a closed circular plasmid, appears to behave just like host chromosomal DNA and it has been demonstrated recently that replication of OriP-containing plasmids is indeed dependent on the chromosomal initiation factors, ORC2 and Cdt1. On the other hand, in the viral productive cycle, the EBV genome is amplified 100- to 1000-fold by the viral replication machinery. EBV productive DNA replication occurs at discrete sites in nuclei, called replication compartments and the lytic programme arrests cell cycle progression and changes the cellular environment greatly. It has been revealed recently that the EBV lytic programme promotes an S-phase like cellular condition, which most favours viral lytic replication. This review describes recent advances regarding the molecular basis of EBV DNA replication during latent and lytic infections and then refers to cellular circumstances after induction of the lytic replication of EBV. Based on the molecular mechanism for the EBV lifestyle, purposeful induction of the lytic form of EBV infection is now advocated as one of the strategies for specific destruction of Epstein-Barr virus (EBV)-associated malignancies where the virus is latently infected.

Monitoring Tumor Recurrence with Nasopharyngeal Swab and Latent Membrane Protein‐1 and Epstein‐Barr Nuclear Antigen‐1 Gene Detection in Treated Patients with Nasopharyngeal Carcinoma

Epstein-Barr virus (EBV) is closely related to nasopharyngeal carcinoma (NPC). Detection of EBV genomic DNA in a nasopharyngeal swab specimen may indicate the presence of NPC, and the EBV genomic DNA is only detected in patients with NPC and not in other head and neck cancers. This study aims to prove that detection of EBV genomic DNA by means of the latent membrane protein (LMP)-1 gene and the Epstein-Barr nuclear antigen (EBNA)-1 gene in the nasopharynx in NPC patients after radiation therapy indicates local recurrence of NPC. Prospective. Nasopharyngeal swab with polymerase chain reaction (PCR)-based LMP-1 and EBNA-1 gene detection was used to monitor local recurrence in 84 NPC patients who completed radiation therapy. Of the 12 patients demonstrating positive LMP-1 and EBNA-1 gene, 11 had local recurrence, and 10 of them had early rT1 mucosal recurrence. Subsequent salvage nasopharyngectomy controlled local disease in nine. Only one local recurrence in the skull base failed to show LMP-1 gene initially. Detection of LMP-1 gene and later verification with EBNA-1 gene from nasopharyngeal swabs in NPC patients after radiation therapy predicted local recurrence with a sensitivity of 91.7% and a specificity of 98.6%. Nasopharyngeal swab with LMP-1 and EBNA-1 gene detection is a useful and reliable method to monitor local recurrence in NPC patients. It helps to detect recurrence early and may improve local control and enhance survival.

Improved In-Gel Competitive Reassociation technique can detect Epstein-Barr virus genome in gastric cancer.

In-gel competitive reassociation (IGCR) technique is one of the DNA subtraction methods with a high capacity. We have improved the IGCR technique and overcame several disadvantages such as time-consuming and complex process with poor reproducibility. The improved IGCR method was applied to the analysis of human gastric adenocarcinoma genomic DNA. The genomic DNA library, which was constructed after the subtraction by IGCR of a normal gastric tissue genomic DNA from an adenocarcinoma genomic DNA in the same patient, contained 13.9% clones of Epstein-Barr virus (EBV) genomic DNA fragment, which is known to be a cause of Burkitt lymphoma. The quantification of EBV genomic DNA revealed that the detection of these EBV fragments originated in the gastric adenocarcinoma genomic DNA was due to the condensation from a few copies to about 100 thousand copies by the improved IGCR technique. This suggests that our improved IGCR technique can efficiently enrich differences between two genomic DNAs with high simplicity.

Screening nasopharyngeal carcinoma by detection of the latent membrane protein 1 (LMP-1) gene with nasopharyngeal swabs.

Nasopharyngeal carcinoma (NPC) is a common head and neck cancer in Taiwan. The goals of the current study were to investigate whether a nasopharyngeal swab technique could provide enough DNA for polymerase chain reaction (PCR) analysis of the Epstein-Barr virus (EBV)-derived latent membrane protein 1 (LMP-1) gene and to determine the feasibility and reliability of diagnosing NPC by detection of LMP-1 in the nasopharynx. 320 adults underwent nasopharyngoscopy and nasopharyngeal swab to obtain cells for the LMP-1 PCR assay; some patients also underwent nasopharyngeal biopsy. An amount of DNA that was sufficient for PCR was extracted from 96.3% of the swab samples. By detecting LMP-1 in nasopharyngeal swabs, NPC was diagnosed with a false positive rate of 12.7% (7 of 55 patients), a false negative rate of 1.6% (4 of 253 patients), sensitivity of 87.3% (48 of 55 patients), specificity of 98.4% (249 of 253 patients), a positive predictive value of 92.3% (48 of 52 patients), and a negative predictive value of 97.3% (249/256 patients). NPC was diagnosed by nasopharyngoscopy with a false positive rate of 38% (30 of 79 patients), a false negative rate of 0.4% (1 of 241 patients), sensitivity of 62% (49 of 79 patients), specificity of 99.6% (240 of 241 patients), a positive predictive value of 98% (49 of 50 patients), and a negative predictive value of 88.9% (240 of 270 patients). Only 7 (0.2%) of 256 patients with a diagnosis other than NPC had LMP-1 detected in the nasopharyngeal space. Detecting EBV genomic LMP-1 by nasopharyngeal swab diagnosed NPC with 87.3% sensitivity and 98.4% specificity. EBV genomic DNA usually is not detected by PCR-based methods in the nasopharyngeal space. Its incidence is estimated to be as low as 0.2% among the general population. The nasopharyngeal swab coupled with PCR-based EBV LMP-1 detection could serve as part of a screening program for high-risk populations.

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.

Presence of Epstein-Barr virus latent membrane protein 1 gene in the nasopharyngeal swabs from patients with nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is the most common head and neck malignancy in southeastern China and Taiwan. Early detection of the local disease followed immediately by proper treatment is essential to increase the cure and survival rates. Because every NPC tumor cell carries Epstein-Barr Virus (EBV) genomes, detection of EBV genomic DNA such as latent membrane protein 1 gene (LMP1) might indicate the presence of NPC. We developed a simple and noninvasive technique of nasopharyngeal swabbing to acquire nasopharyngeal cells for detecting the presence of EBV genome. The aim of this study was to investigate the feasibility and reliability of this technique. We collected nasopharyngeal cells by means of a nasopharyngeal swabbing technique and detected the presence of EBV LMP1 with polymerase chain reaction (PCR). Thirty-eight swab specimens were obtained from patients with NPC who were newly diagnosed or were just beginning radiotherapy. Two groups of control subjects were recruited, including 20 patients with other head and neck cancers and eight family members of the NPC patients. An additional group of 65 NPC patients were enrolled in the course of regular follow-up after definitive radiotherapy. All of the samples yielded sufficient DNA for PCR amplification. Thirty-six of 38 NPC swab samples were positive for EBV LMP1, and all the control subjects had swab sample results negative for EBV. All five patients with suspected local recurrence exhibited positive EBV test results. Demonstration of EBV LMP1 in the nasopharyngeal swab specimens detected NPC with a sensitivity of 94.7% and specificity of 100%. This study confirms the reliability and feasibility of nasopharyngeal swab in the predicting and screening of NPC.

Infection of primary human monocytes by Epstein-Barr virus.

Previous studies have reported that infection of monocytes by viruses such as cytomegalovirus and human immunodeficiency virus weakens host natural immunity. In the present study, we demonstrated the capability of Epstein-Barr virus (EBV) to infect and replicate in freshly isolated human monocytes. Using electron microscopy analysis, we observed the presence of EBV virions in the cytoplasm and nuclei of approximately 20% of monocytes. This was confirmed by Southern blot analysis of EBV genomic DNA sequences in isolated nuclei from monocytes. Infection of monocytes by EBV leads to the activation of the replicative cycle. This was supported by the detection of immediate-early lytic mRNA BZLF-1 transcripts, and by the presence of two early lytic transcripts (BALF-2, which appears to function in DNA replication, and BHRF-1, also associated with the replicative cycle). The late lytic BcLF-1 transcripts, which code for the major nucleocapsid protein, were also detected, as well as EBNA-1 transcripts. However, attempts to detect EBNA-2 transcripts have yielded negative results. Viral replication was also confirmed by the release of newly synthesized infectious viral particles in supernatants of EBV-infected monocytes. EBV-infected monocytes were found to have significantly reduced phagocytic activity, as evaluated by the quantification of ingested carboxylated fluoresceinated latex beads. Taken together, our results suggest that EBV infection of monocytes and alteration of their biological functions might represent a new mechanism to disrupt the immune response and promote viral propagation during the early stages of infection.

Polymerase chain reaction amplification of archival material for epstein-barr virus, cytomegalovirus, human herpesvirus 6, and parvovirus B19 in children with bone marrow hemophagocytosis

Bone marrow hemophagocytosis may occur as an incidental finding, or it may be a manifestation of a systemic and potentially lethal disorder. When systemic, the proliferation is termed hemophagocytic lymphohistiocytosis (HLH), a clinicopathologic entity characterized by a widespread proliferation of benign hemophagocytic histiocytes, fever, pancytopenia, deranged liver function, and frequently coagulopathy and hepatosplenomegaly. A variety of infectious agents, including Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), and parvovirus B19 (PVB19), have been associated with HLH, but the relative frequency of each using one technique has not been evaluated. In addition, infectious causes of incidental bone marrow hemophagocytosis, not occurring in the setting of HLH, have not been evaluated. Review of bone marrow reports from bone marrow examinations done between December 1986 and June 1997 showed that 20 children aged 2 months to 15 years had bone marrow examinations that indicated hemophagocytosis. Archival materials from 19 patients were successfully retrieved, and DNA was extracted from archived unstained coverslips with subsequent polymerase chain reaction for EBV, CMV, HHV6, and PVB19 genomic DNA. DNA extracted from 16 bone marrow specimens of age-matched children was used as negative controls. Eleven of the 19 patients fulfilled the clinical and pathological criteria for HLH; the remaining eight patients had isolated hemophagocytosis without a systemic presentation. Viral DNA was detected in 8 of 11 patients with HLH but in none of eight patients with isolated hemophagocytosis. EBV was present in five of the bone marrows, followed in frequency by HHV6, CMV, and PVB19. Infection with more than one agent was present in three patients. Only one control patient was positive for HHV6 DNA; the remaining control patients were negative for all viruses. Viral infection, detected by PCR analysis of bone marrow, is a common finding in patients with HLH but not in patients with isolated bone marrow hemophagocytosis. This technique may provide another marker to aid in the diagnosis of HLH and suggests a different cause of hemophagocytosis occurring in patients with and without HLH.

Epstein‐Barr virus infection in the neoplastic and nonneoplastic cells of lymphoid malignancies

BACKGROUND The Epstein-Barr virus (EBV) has been frequently detected in lymphoid malignancies. However, EBV infection in the nonneoplastic cells of lymphoid malignancies has not been extensively studied. METHODS Four hundred nine cases of lymphoid malignancies including 377 non-Hodgkin's lymphoma (NHL) and 32 Hodgkin's disease (HD) were examined for EBV infection by EBER-1 in situ hybridization (EBER-ISH), immunostaining against LMP-1, Epstein–Barr nuclear antigen 2 (EBNA2) and ZEBRA, and Southern hybridization using a BamHIW fragment as a probe. Double staining with EBER-ISH and immunostaining against CD20, CD45RO, and LMP-1 was performed in selected cases. RESULTS Although EBER-1-positive cells (EPCs) were detected in 49 of 276 B-cell lymphomas, 31 of 100 T-cell lymphomas, 1 of 1 natural killer-cell lymphoma, and 17 of 32 HDs, almost all of the tumor cells were exclusively EBER-1-positive in the 10 NHL cases. Some EPCs were of different cell lineages than the tumor cells in 15 of the 26 NHLs examined by double staining. LMP-1, EBNA2, and ZEBRA were detected in 22, 4, and 3 cases, respectively. In 4 LMP-1-positive HDs, double staining revealed that some EBER-1-positive Reed-Sternberg cells were negative for LMP-1. EBV genomic DNA was detected in 8 of the 39 examined cases. CONCLUSIONS T-cell lymphomas contained EPCs more frequently than B-cell lymphomas. Nonneoplastic lymphocytes were infected with EBV more frequently than lymphoma cells. Rowe's latency II may be unstable in lymphoid malignancies. Some NHLs, especially T-cell lymphoma, may provide favorable conditions for EBV infection of nonneoplastic lymphocytes. Cancer 1996;77:2339-47.

Epstein-Barr virus infection in the neoplastic and nonneoplastic cells of lymphoid malignancies

The Epstein-Barr virus (EBV) has been frequently detected in lymphoid malignancies. However, EBV infection in the nonneoplastic cells of lymphoid malignancies has not been extensively studied. Four hundred nine cases of lymphoid malignancies including 377 non-Hodgkin's lymphoma (NHL) and 32 Hodgkin's disease (HD) were examined for EBV infection by EBER-1 in situ hybridization (EBER-ISH), immunostaining against LMP-1, Epstein-Barr nuclear antigen 2 (EBNA2) and ZEBRA, and Southern hybridization using a BamHIW fragment as a probe. Double staining with EBER-ISH and immunostaining against CD20, CD45RO, and LMP-1 was performed in selected cases. Although EBER-1-positive cells (EPCs) were detected in 49 of 276 B-cell lymphomas, 31 of 100 T-cell lymphomas, 1 of 1 natural killer-cell lymphoma, and 17 of 32 HDs, almost all of the tumor cells were exclusively EBER-1-positive in the 10 NHL cases. Some EPCs were of different cell lineages than the tumor cells in 15 of the 26 NHLs examined by double staining. LMP-1, EBNA2, and ZEBRA were detected in 22, 4, and 3 cases, respectively. In 4 LMP-1-positive HDs, double staining revealed that some EBER-1-positive Reed-Sternberg cells were negative for LMP-1, EBV genomic DNA was detected in 8 of the 39 examined cases. T-cell lymphomas contained EPCs more frequently than B-cell lymphomas. Nonneoplastic lymphocytes were infected with EBV more frequently than lymphoma cells. Rowe's latency II may be unstable in lymphoid malignancies. Some NHLs, especially T-cell lymphoma, may provide favorable conditions for EBV infection of nonneoplastic lymphocytes.