Epstein-Barr Virus (EBV) Infection by Cocultivation of Fibroblast-Dominant Cell Line and EBV-Transformed Lymphocytes

ORL ◽  
1989 ◽  
Vol 51 (2) ◽  
pp. 69-76
Author(s):  
M. Furukawa ◽  
M. Kamide ◽  
T. Miwa ◽  
M. Sakumoto ◽  
R. Umeda
Keyword(s):  
Cell Line ◽  
Epstein Barr Virus ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr

scholarly journals Signatures of B-cell receptor diversity in B lymphocytes following Epstein-Barr virus transformation

2019 ◽  
Vol 51 (6) ◽  
pp. 197-207
Author(s):  
Meimei Lai ◽  
Qiongdan Wang ◽  
Yutian Lu ◽  
Xi Xu ◽  
Ying Xia ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Epstein Barr Virus ◽  
High Throughput Sequencing ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Human Virus ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr

Epstein-Barr virus (EBV) is a widespread human virus that establishes latent infection, potentially leading to tumors, hematological disorders, and other severe diseases. EBV infections are associated with diverse symptoms and affect various organs; therefore, early diagnosis and treatment are crucial. B cell receptor (BCR) repertoires of B cell surface immunoglobulins have been widely studied for their association with various infectious diseases. However, the specific genetic changes that modulate the BCR repertoires after an EBV infection are still poorly understood. In this study, we employed high-throughput sequencing (HTS) to investigate the diversity of BCR repertoires in an EBV-transformed lymphoblastic cell line (LCL). Compared with the noninfected control B cell line, the LCL exhibited a decrease in overall BCR diversity but displayed an increase in the expansion of some dominant rearrangements such as IGHV4-31/IGHJ4, IGHV4-59/IGHJ4, IGHV5-51/IGHJ3, and IGHV3-74/IGHJ3. A higher frequency of occurrence of these rearrangement types was confirmed in patients with EBV infection. Interestingly, the IGHV3-74 rearrangement was only detected in EBV-infected children, suggesting that our experimental observations were not coincidental. In addition, we identified a highly dominant consensus motif, CAR(xRx)YGSG(xYx)FD, in complementarity-determining region 3 (CDR3) sequences of the heavy chain in the LCL. Our findings demonstrated the utility of HTS technology for studying the variations in signature motifs of the BCR repertoires after EBV infection. We propose that the analysis of BCR repertoire sequences represents a promising method for diagnosing early EBV infections and developing novel antibody- and vaccine-based therapies against such infections.

scholarly journals A Positive Autoregulatory Loop of LMP1 Expression and STAT Activation in Epithelial Cells Latently Infected with Epstein-Barr Virus

2003 ◽  
Vol 77 (7) ◽  
pp. 4139-4148 ◽  
Author(s):  
Honglin Chen ◽  
Lindsey Hutt-Fletcher ◽  
Liang Cao ◽  
S. Diane Hayward
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Barr Virus ◽  
Ebv Infection ◽  
Lmp1 Expression ◽  
Cell Line Hela ◽  
Epstein Barr ◽  
Phosphorylated Stat3

ABSTRACT STAT3 and STAT5 are constitutively activated and nuclear in nasopharyngeal carcinoma (NPC) cells. In normal signaling, STATs are only transiently activated. To investigate whether Epstein-Barr virus (EBV), and in particular the protein LMP1, contributes to sustained STAT phosphorylation and activation in epithelial cells, we examined STAT activity in two sets of paired cell lines, HeLa, an EBV-converted HeLa cell line, HeLa-Bx1, the NPC-derived cell line CNE2-LNSX, and an LMP1-expressing derivative, CNE2-LMP1. EBV infection was associated with a significant increase in the tyrosine-phosphorylated forms of STAT3 and STAT5 in HeLa-Bx1 cells. This effect correlated with LMP1 expression, since phosphorylated STAT3 and STAT5 levels were also increased in CNE2-LMP1 cells relative to the control CNE2-LNSX cells. No change was observed in STAT1 or STAT6 phosphorylation in these cell lines, nor was there a significant change in the levels of total STAT3, STAT5, STAT1, or STAT6 protein. Tyrosine phosphorylation allows the normally cytoplasmic STAT proteins to enter the nucleus and bind to their recognition sequences in responsive promoters. The ability of LMP1 to activate STAT3 was further established by immunofluorescence assays in which coexpression of LMP1 in transfected cells was sufficient to mediate nuclear relocalization of Flag-STAT3 and by an electrophoretic mobility shift assay which showed that LMP1 expression in CNE2-LNSX cells was associated with increased endogenous STAT3 DNA binding activity. In addition, the activity of a downstream target of STAT3, c-Myc, was upregulated in HeLa-Bx1 and CNE2-LMP1 cells. A linkage was established between interleukin-6 (IL-6)- and LMP1-mediated STAT3 activation. Treatment with IL-6 increased phosphorylated STAT3 levels in CNE2-LNSX cells, and conversely, treatment of CNE2-LMP1 cells with IL-6 neutralizing antibody ablated STAT3 activation and c-Myc upregulation. The previous observation that STAT3 activated the LMP1 terminal repeat promoter in reporter assays was extended to show upregulated expression of endogenous LMP1 mRNA and protein in HeLa-Bx1 cells transfected with a constitutively activated STAT3. A model is proposed in which EBV infection of an epithelial cell containing activated STATs would permit LMP1 expression. This in turn would establish a positive feedback loop of IL-6-induced STAT activation, LMP1 and Qp-EBNA1 expression, and viral genome persistence.

scholarly journals Identification of Epstein-Barr Virus (EBV) Nuclear Antigen 2 (EBNA2) Target Proteins by Proteome Analysis: Activation of EBNA2 in Conditionally Immortalized B Cells Reflects Early Events after Infection of Primary B Cells by EBV

2004 ◽  
Vol 78 (8) ◽  
pp. 3941-3952 ◽  
Author(s):  
Martin Schlee ◽  
Tanja Krug ◽  
Olivier Gires ◽  
Reinhard Zeidler ◽  
Wolfgang Hammerschmidt ◽  
...  
Keyword(s):  
B Cells ◽  
Protein Expression ◽  
B Cell ◽  
Cell Line ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Target Proteins ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) is a ubiquitous B-lymphotropic herpesvirus associated with several malignant tumors, e.g., Burkitt's lymphoma and Hodgkin's disease, and is able to efficiently immortalize primary B lymphocytes in vitro. The growth program of infected B cells is initiated and maintained by the viral transcription factor EBV nuclear antigen 2 (EBNA2), which regulates viral and cellular genes, including the proto-oncogene c-myc. In our study, patterns of protein expression in B cells with and without EBNA2 were analyzed by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. For this purpose, we used a conditional immortalization system for EBV, a B cell line (EREB2-5) that expresses an estrogen receptor-EBNA2 fusion protein. In order to discriminate downstream targets of c-Myc from c-Myc-independent EBNA2 targets, we used an EREB2-5-derived cell line, P493-6, in which c-Myc is expressed under the control of a tetracycline-regulated promoter. Of 20 identified EBNA2 target proteins, 11 were c-Myc dependent and therefore most probably associated with proliferation, and one of these proteins was a posttranslationally modified protein, i.e., hypusinylated eIF5a. Finally, to estimate the relevance of EBNA2 targets during early EBV infection, we analyzed the proteomes of primary B cells before and after infection with EBV. The protein expression pattern induced upon EBV infection was similar to that following EBNA2 activation. These findings underscore the value of EREB2-5 cells as an appropriate model system for the analysis of early events in the process of EBV-mediated B-cell immortalization.

Analysis of Epstein-Barr Virus Infections in Lymphoma Cell Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3906-3906
Author(s):  
Cord C. Uphoff ◽  
Sabine A. Denkmann ◽  
Hans G. Drexler
Keyword(s):  
Cell Line ◽  
B Lymphocytes ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Human Populations ◽  
Cell Leukemia ◽  
Linear Dna ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr

Abstract Epstein-Barr virus (EBV, human herpesvirus type 4) is ubiquitously distributed in all human populations, reaching infection rates of more than 90%. EBV is known to infect B- lymphocytes and mucosal epithelium cells and to establish latent or productive infections. The virus is the causative agent of infectious mononucleosis and closely associated with the endemic form of Burkitt lymphoma (BL). EBV has also been associated with various lymphoid and epithelial malignancies, such as Hodgkin, T-cell, and AIDS-related lymphomas, and lymphoepithelioma-like carcinomas of several organs. In vitro, B- lymphocytes are transformed by EBV into permanent lymphoblastoid cell lines (B-LCL). We investigated the EBV infection status of primate cell lines by PCR (406 human, 4 monkey). This method detects EBV genomes integrated into the eukaryotic chromosomes, non-integrated EBV episomes, and linear genomes of active EBV particles. The analyses revealed that 38/410 cell lines contain the EBV genome. All EBV+ cell lines were established from B- lineage leukemia/lymphoma cells (13/52 B-non-Hodgkin cell lines, 10/13 BL cell lines, 2/2 hairy cell leukemia cell lines, 1/6 plasma cell leukemia/myeloma cell lines) or are B-LCLs (9/9), natural killer cells (2/2), and one monkey cell line. No cell lines from other tissues were found to be EBV+. To further examine the production of EBV particles in the PCR+ cell lines, we analyzed the expression of the BZLF1 protein by Western blotting applying a ZEBRA monoclonal antibody. The cell lines were analyzed untreated as well as treated with the phorbol ester TPA for 3 days to induce the lytic phase of the EBV infection. Four cell lines exhibited a BZLF1 specific band a priori; after stimulation with TPA, 4 further cell lines expressed BZLF1 protein to various extents. To distinguish between linear DNA of herpesviruses (DNA form of active viruses) and covalently closed circles of episomal DNA, we performed Gardella gels applying crude lysates from cell cultures. Except for cell line NAMALWA and its subclones, DG-75, DOHH-2, and OCI-LY19 (all EBV-PCR+ cell lines) showed at least one band of episomal genomes. Some cell lines showed two episomal bands pointing to a double infection or to mutated episomes. The amount of linear DNA does not correlate with the number of episomes. Southern blots of genomic DNA revealed different genotypes of EBV, except for those cell lines which were established with B95-8 virus particles. To determine distribution of EBV genomes in single cells, we established a fluorescence in situ hybridization (FISH) method with a Cy3-labeled cosmid clone containing a genomic EBV fragment. The method showed for various cell lines that only a few cells contain high amounts of EBV genomes (several hundred) whereas the vast majority harbors only a few genomes in the nuclei. FISH appears to be superior to other methods, allowing for EBV analysis at the single cell level to determine the cellular permissiveness. In summary, we could show that EBV is constitutively produced in a few B-lymphoma derived cell lines and can be induced in several other cell lines. These cell lines represent valuable tools for further investigation into the biology of EBV infection.

scholarly journals In vitro Studies and Clinical Observations Imply a Synergistic Effect Between Epstein-Barr Virus and Dengue Virus Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiao-Mei Deng ◽  
Ling-Zhai Zhao ◽  
Xue-Ying Liang ◽  
Dan Li ◽  
Lei Yu ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Dengue Virus ◽  
Cell Line ◽  
Epstein Barr Virus ◽  
Severe Dengue ◽  
Complex Spectrum ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr

Dengue virus (DENV) infection can lead to a complex spectrum of clinical outcomes, ranging from asymptomatic infection to life-threatening severe dengue. The reasons for thus drastically varying manifestations of the disease remain an enigma. Herein, we reported an original discovery of the synergistic effect between preexisting Epstein–Barr virus (EBV) infection and DENV superinfection in vitro and of a strong correlation of these two viruses in the clinical samples from dengue patients. We showed that (I) DENV-2 infection of an EBV-positive cell line (EBV + Akata cell) reactivated EBV, and it could be blocked by wortmannin treatment. (II) Examination of human peripheral blood mononuclear cell (PBMC) samples from dengue patients revealed significantly elevated cell-associated EBV DNA copy number at the time of hospitalization vs. at the time of disease recovery in most individuals. (III) EBV infection promoted DENV propagation in both EBV-hosting B cells and indirectly in THP-1 cells, supported by the following evidence: (A) EBV + Akata cells were more permissive to DENV-2 infection compared with Akata cells harboring no EBV virus (EBV- Akata cells). (B) Low-molecular weight fraction secreted from EBV + Akata cells could enhance DENV-2 propagation in monocytic THP-1 cells. (C) While reactivation of EBV in EBV + Akata cells further increased DENV-2 yield from this cell line, pharmacological inhibition of EBV replication by acyclovir had the opposite effect. To our knowledge, this is the first investigation demonstrating a positive correlation between EBV and DENV in vitro and in human biospecimens.

scholarly journals Air-Liquid Interface Method To Study Epstein-Barr Virus Pathogenesis in Nasopharyngeal Epithelial Cells

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
Author(s):  
Elizabeth A. Caves ◽  
Sarah A. Cook ◽  
Nara Lee ◽  
Donna Stoltz ◽  
Simon Watkins ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Infected Cell ◽  
Cell Line ◽  
Epstein Barr Virus ◽  
Culture Method ◽  
Liquid Interface ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr ◽  
Air Liquid Interface

ABSTRACT Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that establishes a latent reservoir in peripheral B-lymphocytes with sporadic reactivation. EBV also infects epithelial cells, predominantly resulting in a lytic infection, which may contribute to EBV transmission from saliva. In the nasopharynx, EBV infection can lead to the clonal expansion of a latently infected cell and the development of nasopharyngeal carcinoma (NPC). The mechanisms governing EBV pathogenesis in nasopharyngeal epithelium are largely unknown. An advanced understanding would depend on a physiologically relevant culture model of polarized airway epithelium. The recent application of the organotypic raft culture in keratinocytes has demonstrated great promise for the use of polarized cultures in the study of EBV permissive replication. In this study, the adaptation of an air-liquid interface (ALI) culture method using transwell membranes was explored in an EBV-infected NPC cell line. In the EBV-infected NPC HK1 cell line, ALI culture resulted in the completion of EBV reactivation, with global induction of the lytic cascade, replication of EBV genomes, and production of infectious progeny virus. We propose that the ALI culture method can be widely adopted as a physiologically relevant model to study EBV pathogenesis in polarized nasal epithelial cells. IMPORTANCE Lifting adherent cells to the air-liquid interface (ALI) is a method conventionally used to culture airway epithelial cells into polarized apical and basolateral surfaces. Reactivation of Epstein-Barr virus (EBV) from monolayer epithelial cultures is sometimes abortive, which may be attributed to the lack of authentic reactivation triggers that occur in stratified epithelium in vivo. In the present work, the ALI culture method was applied to study EBV reactivation in nasopharyngeal epithelial cells. The ALI culture of an EBV-infected cell line yielded high titers and can be dissected by a variety of molecular virology assays that measure induction of the EBV lytic cascade and EBV genome replication and assembly. EBV infection of polarized cultures of primary epithelial cells can be challenging and can have variable efficiencies. However, the use of the ALI method with established EBV-infected cell lines offers a readily available and reproducible approach for the study of EBV permissive replication in polarized epithelia.

Sign in / Sign up

Export Citation Format

Share Document