OBJECTIVETo analyze the characteristic of nuclear antigen 1 gene and latent membrane protein 1 gene of Epstein-Barr virus in primary EBV infection in children in Beijing area in 2005-2012.

METHODSPolymerase chain reaction (PCR) was used to amplify the EBNA-3C, EBNA1 and LMP1 genes. The amplified products were sequenced directly and the sequences were analyzed by BioEdit 7. 0. 9 and MEGA 4. 0. 2.

RESULTSType A EBV was detected in 98% samples. Nucleotide sequence analysis of the carboxy-terminal region of EBNA1 showed that Vvvl was deteted in 98% samples. DNA sequence analysis of LMP1 C-terminus indicated that China 1 was 90% in this study. There were no significant differences in the frequency of Vvv1 and China 1 between the IM and HLH samples (P = 1.00). Linkage analysis of EBV types, EBNA1 and LMP1 variants indicated that 90% of EBV type A was associated with EBNA1-Vvv1 variant and LMP1-China 1 variant in 40 cases. Full length of LMP1 gene was successfully amplified in 35 cases. Four Chinese groups (CG1-4) were identified. The percentage of CG1-CG4 were 85%, 6%, 6% and 3%, respectively.

CONCLUSIONEBV type A is predominant in primary EBV infection in children in Beijing Area. EBNA1-Vvv1 and LMP1-China 1 variants were predominant genotypes in this area. There is a high linkage between EBNA1-Vvv1 variant and LMP1-China 1 variant. Four Chinese groups (CG1-4) were identified according to the full length of LMP1 gene and CG1 was the most prevalent.

China ; Epstein-Barr Virus Infections ; virology ; Epstein-Barr Virus Nuclear Antigens ; genetics ; Genetic Linkage ; Herpesvirus 4, Human ; classification ; genetics ; Humans ; Time Factors ; Viral Matrix Proteins ; genetics

Latent Epstein-Barr virus (EBV) infection has a substantial role in causing many human disorders. The persistence of these viral genomes in all malignant cells, yet with the expression of limited latent genes, is consistent with the notion that EBV latent genes are important for malignant cell growth. While the EBV-encoded nuclear antigen-1 (EBNA-1) and latent membrane protein-2A (LMP-2A) are critical, the EBNA-leader proteins, EBNA-2, EBNA-3A, EBNA-3C and LMP-1, are individually essential for in vitro transformation of primary B cells to lymphoblastoid cell lines. EBV-encoded RNAs and EBNA-3Bs are dispensable. In this review, the roles of EBV latent genes are summarized.
Epstein-Barr Virus Infections/complications/virology ; Epstein-Barr Virus Nuclear Antigens/genetics/metabolism ; *Genes, Viral ; Herpesvirus 4, Human/*physiology ; Humans ; MicroRNAs/genetics ; Neoplasms/etiology ; Protein Binding ; RNA, Viral/genetics ; Viral Matrix Proteins/genetics/metabolism ; *Virus Latency

This study aimed to construct recombinant adenovirus expressing Epstein-Barr nuclear antigen 1 (EBNA1) against nasopharyngeal carcinoma (NPC). The C-terminal region fragment of the ebna1 gene of Epstein-Barr virus was amplified from the standard strain B95-8 by polymerase chain reaction (PCR). The gene fragment was inserted into the pDC316 shuttle plasmid using the EcoRI and BgIII restriction enzyme sites. The pDC316-ebna1 shuttle plasmid and pBHG helper plasmid were cotransfected into HEK293 cells after sequencing. The soluble protein was extracted from HEK293 cells, which caused apparent cytopathic effects. The transcription and expression of the ebna1 gene were confirmed using flow cytometry and Western blotting. rAd-ebna1 titers were measured by the TCID50. rAd-ebna1 was injected into BALB/c mice at a dose of 2 x 10(8) VP per mouse, EBNA1 epitope-specific responses were measured at 1st, 2nd, 4th and 8th weeks post-immunization. The target fragment of ebna1 (939 bp) was obtained by PCR, and was in consensus with the sequence from the standard strain B95-8. Cytopathic effects were observed after the pDC316-ebna1 shuttle plasmid and pBHG helper plasmid were cotransfected into HEK293 cells. rAd-ebna1 was successfully recombined in HEK293 cells. EBNA1 protein was detected in HEK293 cells, rAd-ebna1 titers reached 10(8) TCID50/mL. Specific responses to CD4+ epitopes of EBNA1 were detected in the immunized mice. In conclusion, rAd-ebna1 was successfully constructed and induced specific responses to CD4+ epitopes of EBNA1 in immunized mice.
Adenoviridae ; genetics ; immunology ; Animals ; CD4-Positive T-Lymphocytes ; immunology ; virology ; Epstein-Barr Virus Infections ; immunology ; prevention & control ; virology ; Epstein-Barr Virus Nuclear Antigens ; administration & dosage ; genetics ; immunology ; Genetic Vectors ; genetics ; immunology ; Herpesvirus 4, Human ; genetics ; immunology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Viral Proteins ; administration & dosage ; genetics ; immunology

The nonviral gene delivery systems are usually not very effective in transferring gene into target cells, and the intensity and duration of the gene expression is very poor. The EBNA1/oriP maintain EBNA1/oriP-based plasmids as episome, contribute to nuclear transport of the plasmid and transcriptional up-regulation of target gene. The EBNA1/oriP based plasmid enhances the transfection rate as well as magnitude and longevity of gene expression. This article reviews recent preclinical gene therapy studies with the EBV plasmid vectors conducted against various diseases. For gene therapy against malignancies, the EBNA1/ oriP based plasmid encoding the HSV1-TK suicide gene was combined with a cationic polymer to transfer into HCC cell line. The expression level of TK gene was 100- to 1000-fold higher than the conventional plasmid. The sensitivity of HCC to ganciclovir (GCV) elevated several hundred-fold. The EBNA1/oriP based plasmid equipped with tumor specific promoter, such as CEA promoter, enabled targeted killing of CEA-positive tumor cell. Transfection of EBNA1/oriP based plasmid carrying IL-12 and IL-18 gene either locally, or systemically, induced therapeufic antitumor immune responses including augmentation of the cytotoxic T lymphocyte and natural killer activities and growth retardation of tumors. For gene therapy of congenital diseases and chronic diseases, the EBNA1/oriP based plasmid encoding the adenosine deaminase gene was transfered into human hematopoietic progenitor cells. The ADA activity was elevated 1.5-to 2-fold. Intracardiomuscrlar transfer of the EBNA1/oriP based plasmid encoding the beta-AR gene may be useful for the treatment of severe heart failure. Human tumor necrosis factoralpha (hTNFalpha) is one of the most important inflammatory cytokines. It has been implicated in many autoimmune and inflammatory diseases. sTNFR can efficiently neutralize the bioactivities of hTNFalpha. In primary study we cloned the chimeric protein sTNFR II-IgG Fc and expect to use it in the gene therapy of the inflammatory disease relative to TNF. In summary, The EBNA1/oriP based plasmid shows advantage in gene therapy of cancer, congenital and inflammatory diseases. Moreover, the EBNA1/oriP element may greatly contribute to the engineering of a human artificial chromosome, the ultimate device for controllable gene therapy.
Epstein-Barr Virus Nuclear Antigens ; genetics ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Herpesvirus 4, Human ; genetics ; metabolism ; Humans ; Muscular Dystrophy, Duchenne ; therapy ; Neoplasms ; therapy ; Plasmids ; genetics ; Replication Origin ; genetics ; Transcription, Genetic ; Transfection ; methods

OBJECTIVETo study the effect of Epstein-Barr virus nuclear antigen 1 (EBNA1) on cell proliferation and cell cycle in nasopharyngeal carcinoma (NPC) cells.

METHODSRecombinant lentivirus that encoded EBNA1 short hairpin RNA (shRNA) was prepared. The C666-EBNA1 (CE) cells were transduced with lentivirus and selected by fluorescence activated cell sorting (FACS) to repress EBNA1 expression. The protein expression levels of EBNA1 were examined by Western blot. The effect of EBNA1 silence on cell proliferation was analyzed by MTT assay and cell growth assay, respectively. Cell cycle was assessed by flow cytometry. The mRNA and protein levels of cell cycle regulators were examined by real-time PCR and Western blot.

RESULTSRecombinant lentivirus encoded EBNA1 shRNA was successfully constructed. The EBNA1 expression in CE cells was significantly reduced by lentivirus-mediated RNA interference. The results of cell counting and MTT assay showed that EBNA1 down-regulation significantly inhibited cell growth in CE-shRNA EBNA1 cells (P < 0.05). Compared with the control group, the percentage of cells in G0-G1 phase was increased from (62.43 ± 6.62)% to (89.66 ± 0.64)% (t = -7.091, P = 0.002), and that in S phase was decreased from (34.93 ± 7.36)% to (7.82 ± 2.44)% (t = 6.095, P = 0.004). The mRNA expressions of c-myc, CDK4, CDK6 and pRb were decreased by 65.60%, 34.06%, 41.05% and 55.29% respectively with the similar results in protein expression levels.

CONCLUSIONSSuppression of EBNA1 may inhibit the growth of nasopharyngeal carcinoma cells in vitro and induce a G1-phase cell cycle arrest, which might be mediated by down-regulation of c-myc, CDK4, CDK6 and pRb.

Carcinoma ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Epstein-Barr Virus Nuclear Antigens ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; Lentivirus ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; pathology ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Transduction, Genetic

Different cytokines are needed in the course of culturing cells to do adoptive immunotherapy. This study was aimed to investigate the differentiation directions of lymphocytes and related gene expression characteristics after combined stimulation of lymphocytes by different cytokines or EBV antigen peptide combined with cytokines. The experiment was divided into 4 groups. The levels of total T lymphocytes (CD3(+)), T helper lymphocytes (CD3(+)CD4(+)), cytotoxic T-lymphocyte (CD3(+)CD8(+)), memory T cells (CD3(+)CD8(+)CD45RO(+)), naive T cells (CD3(+)CD8(+)CD45RA(+)), Th2 cells (CD3(+)CD30(+)), B cells (CD19(+)), NK cells (CD56(+)), naive T regulatory cells (CD4(+)CD25(+)), precise T regulatory cells (CD4(+)CD25(+)FOXP3(+)) were detected by flow cytometry. The expression levels of house-keeping gene (mad1, pten), T helper cells transcriptional regulatory gene t-bet (Th1), gata3 (Th2), cytokine IFN-γ(Th1), IL-4(Th2) were detected by using RT-PCR. The results showed that CTL in EBV polypeptide group were dominant cells with certain clinical effects. Comparison of result of EBV polypeptide group with other 3 different cytokine stimulating groups demonstrated that EBV antigen peptide had much more effects on stimulating CTL generation. The expression of IFN-γ gene was significantly increased; the T helper differentiation-related gene t-bet, gata3 also increased evidently, while expression change of house-keeping gene mad1 and pten were not evident. Addition of different cytokines and antigen peptides in culture may be much more effective on stimulating CTL generation. It is concluded that specific CTL can be obtained by using the lymphocytes co-cultured with EBV and cytokines, and the different cytokines play different roles in cell differentiation.
Cells, Cultured ; Cytokines ; immunology ; metabolism ; Epstein-Barr Virus Nuclear Antigens ; genetics ; Flow Cytometry ; Humans ; Immunotherapy, Adoptive ; Lymphocyte Count ; Lymphoma, Extranodal NK-T-Cell ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo understand Epstein-Barr virus (EBV) infection of gastric carcinoma cells.

METHODSThe authors tested the infection of a signet ring cell line HSC-39 derived from human gastric carcinoma with Akata and P3HR-1 strains of EBV. Akata and P3HR-1 infected of EBV cell clones were isolated by a limiting dilution method.

RESULTSEBV-encoded small RNAs (EBERs) were expressed in the infected cells with each EBV strain by in situ hybridization. The EBV infected parental cells and most clones expressed EBNA1, but not EBNA2, latent membrane protein (LMP) 1 and LMP2A. Both EBV strains infected parental cells and clones presented type I latency. The uninfected HSC-39 cells were negative for CD21 expression; however, the Akata but not P3HR-1-infected clones were positive for CD21 expression at mRNA level.

CONCLUSIONThese results demonstrated that EBV infecting HSC-39 by CD21-independent pathway. This study also defined a signet ring cell line as a new target for EBV.

Carcinoma, Signet Ring Cell ; pathology ; virology ; Cell Line, Tumor ; Epstein-Barr Virus Nuclear Antigens ; analysis ; Herpesvirus 4, Human ; genetics ; physiology ; Humans ; RNA, Messenger ; RNA, Viral ; analysis ; Receptors, Complement 3d ; analysis ; genetics ; physiology ; Stomach Neoplasms ; pathology ; virology

Methylation of p16 is an important mechanism in cervical carcinogenesis. However, the relationship between cervical squamous cell carcinoma (SCC) and Epstein-Barr virus (EBV) remains controversial. Here, we explored whether EBV infection and/or p16 gene inactivation would play any role in cervical carcinogenesis. Eighty-two specimens included 41 invasive SCCs, 30 cervical intraepithelial neoplasm (CIN; CIN 1, 11 cases, CIN II, 3 cases, CIN III 16 cases) and 11 nonneoplastic cervices. EBV was detected by polymerase chain reaction (PCR) for EBNA-1 and in situ hybridization for EBER-1. The p16 methylation-status and the expression of p16 protein were studied by methylation-specific PCR and immunohistochemistry, respectively. The materials were divided into four groups: 1) nonneoplastic cervices, 2) CIN I, 3) CIN II-III and 4) invasive SCCs. p16 methylation and p16 immunoexpressions increased in CIN and invasive SCCs than nonneoplastic tissue. p16-methylation and p16-immunoreactivities were higher in the EBV-positive group (p=0.009, p<0.001) than in the EBV-negative group. EBV was detected more frequently in CIN and SCCs than nonneoplastic cervices. In conclusion, a correlation between p16 methylation, p16 immunoreactivity and the detection of EBV strongly suggested that the cooperation of EBV and p16 gene may play a synergic effect on cell cycle deregulation.
Carcinoma, Squamous Cell/genetics/*pathology/virology ; Comparative Study ; Cyclin-Dependent Kinase Inhibitor p16/analysis/*genetics ; *DNA Methylation ; DNA, Viral/genetics/isolation & purification ; Epstein-Barr Virus Infections/genetics/*pathology/virology ; Epstein-Barr Virus Nuclear Antigens/genetics ; Female ; Herpesvirus 4, Human/genetics ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Polymerase Chain Reaction ; Precancerous Conditions/genetics/*pathology/virology ; RNA, Viral/genetics ; Research Support, Non-U.S. Gov't ; Uterine Cervical Neoplasms/genetics/*pathology/virology