DNA Replication ; physiology ; Epstein-Barr Virus Infections ; virology ; Herpesvirus 4, Human ; genetics ; metabolism ; physiology ; Humans

OBJECTIVETo study the genetic stability of an immortalized cell line transformed by Epstein-Barr virus (EBV) after long subculture process.

METHODSIn the present study, the genetic stability including chromosome diploidy, karyotypes and microsatellite DNA were evaluated with chromosome banding techniques and microsatellite DNA detection. The telomerase activity of the immortalized cell line was detected by using the telomerase assay kit.

RESULTSFrom passage 1 to 30, there were no change of the diploidy, karyotypes of chromosome and microsatellite DNA, and the telomerase activity is negative.

CONCLUSIONThis study indicates that the immortalized cell line remains stable genetically within limited passages.

Cell Transformation, Viral ; genetics ; Herpesvirus 4, Human ; genetics ; Humans ; Lymphocytes ; cytology ; metabolism ; virology ; Microsatellite Repeats ; genetics ; Polymerase Chain Reaction

OBJECTIVETo elucidate the regulation of the phosphorylation of epidermal growth factor receptor (EGFR) by the EB virus encoded latent membrane protein 1 (LMP1) in nasopharyngeal carcinoma cell line.

METHODSThe levels of EGFR expression and phosphorylation in pTet-on LMP1 HNE2 cell, a nasopharyngeal carcinoma (NPC) cell line, in the dynamic expression of LMP1 induced by different concentrations of doxycycline (Dox) were observed. The EGFR dominant negative mutant and LMP1 antisense expression plasmid were transiently transfected into pTet-on LMP1 HNE2 cells by lipofectamine, and the changes in EGFR phosphorylation were observed by immunocoprecitation and Western blot. The changes in EGFR phosphorylation were observed after EGF treatment.

RESULTSIn pTet-on LMP1 HNE2 cells, Dox-induced LMP1 upregulated EGFR expression and phosphorylation in a dose-dependent manner. After EGFR dominant negative mutant was transfected into pTet-on LMP1 HNE2 cells, the increase of EGFR phosphorylation was inhibited completely. When LMP1 antisense expression plasmid was transfected into pTet-on LMP1 HNE2 cells, the levels of EGFR phosphorylation were also inhibited significantly. Meanwhile, after EGF had been added into pTet-on LMP1 HNE2 cells, increase of EGFR phosphorylation was induced, but it was completely blocked by EGFR dominant negative mutant and the introduction of LMP1 antisense.

CONCLUSIONEB virus encoded LMP1 not only induces the dose-dependent expression of EGFR, but also the dose-dependent phosphorylation of EGFR. The phosporylation of EGFR may play a vital role in the development of nasopharyngeal carcinoma.

Blotting, Western ; Epidermal Growth Factor ; metabolism ; Herpesvirus 4, Human ; metabolism ; Humans ; Nasopharyngeal Neoplasms ; pathology ; virology ; Phosphorylation ; Receptor, Epidermal Growth Factor ; metabolism ; Tumor Cells, Cultured ; Viral Matrix Proteins ; metabolism

The Epstein-Barr virus (EBV) is a gamma herpes virus associated with several types of malignancies. The EBV encodes viral microRNAs (miRNAs) that can target genes within cells. The EBV participates in signal transduction as well as the proliferation and differentiation of cells. How the target genes and functions of EBV-encoded miRNAs contribute to the pathogenesis of EBV is an important research topic. Some target genes have been validated since EBV-encoded miRNAs were discovered and, in this article, we summarize them and their functions.
Animals ; Epstein-Barr Virus Infections ; genetics ; metabolism ; virology ; Herpesvirus 4, Human ; genetics ; physiology ; Humans ; MicroRNAs ; genetics ; metabolism ; RNA, Viral ; genetics ; metabolism

OBJECTIVEEpstein-Barr virus (EBV) is a common causative agent of infectious mononucleosis (IM) and capable of efficiently immortalizing primary B cells into continuously growing lymphoblastoid cells in vitro. As B cell activation antigen, CD23 expression is induced by EBV infection of B cells and remains constitutively expressed at high levels in virtually all EBV-immortalized cells, which have been strongly linked to the development of B-cell lymphoproliferative disease and lymphoma. Whereas previous studies were performed in vivo in animals or ex vivo cultures, the present study aimed to explore the role of EBV-immortalized cells (CD23(+)/CD19(+)) in vivo analysis of children with EBV-IM.

METHODSIn a prospective trial, a group of 30 patients with IM (18 boys and 12 girls) with mean age of 3.9 +/- 1.3 years (range 6 months to 8 years) were enrolled. Clinical diagnosis of IM was confirmed based on fever, lymphadenopathy, splenomegaly, lymphocytosis (> 50%), atypical lymphocytes (> 10%) in blood smears and the elevated levels of IgM antibody against EBV capsid antigen. The day of onset of fever was recognized as day 1 of illness. Blood samples taken during acute (3 - 5 days), early convalescent (about 11 - 15 days) and convalescent phase (about 30 - 45 days) were analyzed for expressions of CD19(+)/CD23(+), CD23, CD19 on peripheral blood mononuclear cells by flow cytometry (FCM) and was compared with those of control group.

RESULTS(1) The levels of CD23(+)/CD19(+) and CD23 expressions were markedly decreased in acute stage [CD23(+)/CD19(+) (2.22 +/- 1.47)%, (132 +/- 91)/mm(3); CD23 (3.12 +/- 1.88)%, (195 +/- 102)/mm(3)] and in early convalescent stage [CD23(+)/CD19(+) (4.51 +/- 2.25)%, (166 +/- 85)/mm(3); CD23 (5.55 +/- 2.76)%, (231 +/- 130)/mm(3)] in patients with IM as compared with those of the healthy controls [CD23(+)/CD19(+) (6.71 +/- 2.25)%, (215 +/- 68)/mm(3); CD23 (7.85 +/- 3.09)%, (249 +/- 86)/mm(3), respectively]. The earlier the history was, the lower the expressive levels were. The levels of CD23(+)/CD19(+) expressions returned to, but those of CD23 expressions exceeded, normal level in convalescent stage [CD23(+)/CD19(+) (6.72 +/- 2.16)%, (213 +/- 108)/mm(3); CD23 (9.46 +/- 2.73)%, (366 +/- 200)/mm(3)]. (2) There was a positive correlation in the expressions of CD23(+)/CD19(+) and CD23 among the three stages (P < 0.01). The positive correlation between the expressions of CD23(+)/CD19(+) and CD19 only occurred during acute stage (P < 0.01). There was no correlation between the expressions of CD23 and CD19 (P > 0.05).

CONCLUSIONEBV-immortalized cells and CD23(+) cells were inhibited effectively during the acute and early convalescent stage of IM. With the recovery of the disease, they gradually recovered and the levels of CD23 expressions exceeded normal level in convalescent stage.

B-Lymphocytes ; metabolism ; Cell Culture Techniques ; Cell Survival ; Child ; Child, Preschool ; Female ; Herpesvirus 4, Human ; pathogenicity ; Humans ; Infant ; Infectious Mononucleosis ; metabolism ; Male ; Receptors, IgE ; metabolism

PURPOSE: The aim of the present study was to evaluate the clinical characteristics of the primary Epstein-Barr virus (EBV) hepatitis with elevation of both serum alkaline phosphatase (ALP) and gamma-glutamyltransferase (gamma-GT) levels in children. MATERIALS AND METHODS: A retrospective study was performed by reviewing of the medical records of 36 patients who were diagnosed with primary EBV hepatitis. The patients were divided into 2 groups: patients with elevated serum ALP and gamma-GT levels (group 1) and patients without (group 2). RESULTS: The classic features of infectious mononucleosis (fever, pharyngitis and/or tonsillitis, and cervical lymphadenitis) were seen in 20 (57.1%) of group 1 patients and 18 (50.0%) of group 2 patients. Hepatitis with elevated serum ALP and gamma-GT levels were present in 14 (38.9%) of the all patients. Of these patients, Jaundice occurred in only 2 (5.6%). The mean levels of aspartate aminotransferase and alanine aminotransferase (ALT) as well as the number of patients with ALT greater than 400 IU/L were significantly different between the groups (177 IU/L vs. 94 IU/L, 418 IU/L vs. 115 IU/L, and 50.0% vs. 13.6%; p=0.001, p=0.001, p=0.026, respectively). The mean duration of elevated serum ALT levels was 17.5 days in group 1 and 9.0 days in group 2 (p=0.013). All patients recovered fully without any chronic or serious complications. CONCLUSION: Primary EBV hepatitis with predominant biochemical abnormalities of the elevation of ALP and gamma-GT is frequent and mostly anicteric. This may represent a benign disease, but a delay in recovery of liver function as well.
Alkaline Phosphatase/genetics/*metabolism ; Child ; Child, Preschool ; Female ; Hepatitis/*enzymology/*pathology/virology ; Herpesvirus 4, Human/*pathogenicity ; Humans ; Infant ; Male ; gamma-Glutamyltransferase/genetics/*metabolism

Antigens, Viral ; genetics ; metabolism ; Cell Line ; Cell Phone ; Gene Expression Regulation, Viral ; radiation effects ; Herpesvirus 4, Human ; physiology ; radiation effects ; Humans ; Immunohistochemistry ; Viral Proteins ; genetics ; metabolism

The incidence of posttransplantation lymphoproliferative disorders (PTLDs) has increased in recent years. Although rare, various types of T-cell lymphoma have been reported and their association with Epstein-Barr virus (EBV) has been compared with B-cell PTLDs. We report a case of splenic peripheral T-cell lymphoma occurring in a 47-yr-old male patient 7 yr after renal allograft transplantation. The spleen showed sinusoidal proliferation of focal CD30 positive, large, atypical lymphoid cells. Positivity for CD3 and cytolytic granule-associated proteins was also demonstrated in the tumor cells, while anaplastic large cell lymphoma kinase (ALK) and CD8 were not expressed. Strong nuclear signals for EBV mRNA were noted by EBER1 in situ hybridization. A molecular genetic study demonstrated a rearrangement of the gamma T-cell receptor gene. To our knowledge, this case is unique in terms of a posttransplant T-cell lymphoma that shows focal CD30, cytolytic granule-associated proteins, and EBV positivity.
Antigens, CD30/genetics ; Antigens, CD30/metabolism ; Herpesvirus 4, Human/genetics ; Herpesvirus 4, Human/metabolism* ; Human ; Kidney Transplantation* ; Lymphoma, T-Cell, Peripheral/pathology ; Lymphoma, T-Cell, Peripheral/virology* ; Male ; Membrane Proteins/metabolism ; Middle Aged ; RNA, Viral ; RNA-Binding Proteins/metabolism ; Serine Endopeptidases/metabolism ; Splenic Neoplasms/pathology ; Splenic Neoplasms/virology*

BACKGROUND: Epstein-Barr virus (EBV) is associated with various lymphoproliferative disorders and nasopharyngeal carcinoma. Recently, some gastric cancer cells were observed to contain the EBV sequence. We detected EBV in gastric cancer by using PCR to determine the frequency of EBV-associated gastric cancer, and performed immunohistochemical staining for the latent membrane protein (LMP1), p53 and CD44 to investigate the possible mechanism in EBV-associated gastric cancer. METHODS: Eighty-seven formalin-fixed and paraffin-embedded blocks (40 gastric adenocarcinomas, 34 adjacent normal tissues, 13 metastatic lymph nodes) from 40 surgically resected gastric specimens were studied. All patients were diagnosed with gastric cancer at the Kang-Nam St. Mary's Hospital between April 1995 and April 1997. After DNA was extracted from each paraffin block, we performed PCR and immunohistochemical staining for the LMP1, p53 and CD44. RESULTS: EBV was detected in 4 of 40 cases (10%). In 1 of 4 EBV-positive cases, EBV was also detected in a metastatic lymph node. The immunohistochemical staining for the LMP1, p53 and CD44 were negative in all the EBV-positive cancer patients. Of the patients having these cancers, 2 had a poorly differentiated adenocarcinoma with a lymphoepithelioma-like morphology. DISCUSSION: The frequency of EBV-associated gastric cancer is about 10% in Korea. Considering the negative result of the immunohistochemical staining for the LMP1, p53 and CD44, EBV-associated gastric cancer seems to have a different mechanism of tumorigenesis from ordinary gastric cancer or other EBV-associated cancers. This specific mechanism must be determined by further large scale studies.
Adenocarcinoma/metabolism/*virology ; Adult ; Antigens, CD44/metabolism ; Female ; Herpesvirus 4, Human/*isolation & purification ; Human ; Immunohistochemistry ; Male ; Middle Aged ; Polymerase Chain Reaction ; Protein p53/metabolism ; Stomach Neoplasms/metabolism/*virology ; Viral Matrix Proteins/metabolism

There has been increasing interest in standardized and quantitative Epstein-Barr virus (EBV) DNA testing for the management of EBV disease. We evaluated the performance of the Real-Q EBV Quantification Kit (BioSewoom, Korea) in whole blood (WB). Nucleic acid extraction and real-time PCR were performed by using the MagNA Pure 96 (Roche Diagnostics, Germany) and 7500 Fast real-time PCR system (Applied Biosystems, USA), respectively. Assay sensitivity, linearity, and conversion factor were determined by using the World Health Organization international standard diluted in EBV-negative WB. We used 81 WB clinical specimens to compare performance of the Real-Q EBV Quantification Kit and artus EBV RG PCR Kit (Qiagen, Germany). The limit of detection (LOD) and limit of quantification (LOQ) for the Real-Q kit were 453 and 750 IU/mL, respectively. The conversion factor from EBV genomic copies to IU was 0.62. The linear range of the assay was from 750 to 10⁶ IU/mL. Viral load values measured with the Real-Q assay were on average 0.54 log₁₀ copies/mL higher than those measured with the artus assay. The Real-Q assay offered good analytical performance for EBV DNA quantification in WB.
DNA, Viral/*blood/metabolism ; Epstein-Barr Virus Infections/diagnosis/virology ; Herpesvirus 4, Human/*genetics/isolation & purification ; Humans ; Limit of Detection ; Reagent Kits, Diagnostic ; Real-Time Polymerase Chain Reaction