Different Strains Of Epstein-Barr Virus Research Articles

A Molecular Basis for the Interplay between T Cells, Viral Mutants, and Human Leukocyte Antigen Micropolymorphism

Mutations within T cell epitopes represent a common mechanism of viral escape from the host protective immune response. The diverse T cell repertoire and the extensive human leukocyte antigen (HLA) polymorphism across populations is the evolutionary response to viral mutation. However, the molecular basis underpinning the interplay between HLA polymorphism, the T cell repertoire, and viral escape is unclear. Here we investigate the T cell response to a HLA-B*35:01- and HLA-B*35:08-restricted (407)HPVGEADYFEY(417) epitope from Epstein-Barr virus and naturally occurring variants at positions 4 and 5 thereof. Each viral variant differently impacted on the epitope's flexibility and conformation when bound to HLA-B*35:08 or HLA-B*35:01. We provide a molecular basis for understanding how the single residue polymorphism that discriminates between HLA-B*35:01/08 profoundly impacts on T cell receptor recognition. Surprisingly, one viral variant (P5-Glu to P5-Asp) effectively changed restriction preference from HLA-B*35:01 to HLA-B*35:08. Collectively, our study portrays the interplay between the T cell response, viral escape, and HLA polymorphism, whereby HLA polymorphism enables altered presentation of epitopes from different strains of Epstein-Barr virus.

Epstein-barr virus diversity in immunocompetent healthy persons: Reassessment of the distribution of genetic variants

Epstein-Barr virus (EBV) has many strains; however, it remains unclear whether a causal relationship exists between different regions and viral genetic variants in healthy persons. This study was designed to examine the relationship between EBV strains in tonsils and adenoids and peripheral blood lymphocytes of the same individuals using different measurements of EBV strain polymorphism. This study examined whether EBV contains two or three copies of a tandem repeat sequence in the first intron of the BZLF-1 gene. The genotype of the virus from P3HR-1, designated Z*, yielded a 415-bp product, and this was distinguished from the smaller, 386-bp product obtained with the B95-8 virus, designated the Z genotype. Simultaneous sequence infections with Z and Z* genotypes were also detected in one of the tonsils examined, suggesting that more than one strain or variant of EBV genotype may be present in a specimen from the same subject. Co-infection with Z and Z* was recognized in two subjects, so variation of the EBV gene may be seen in at least two different strains of EBV. It was seen that Z and Z* strain-infected cells are constantly in flux through lymph nodes and/or the blood stream in healthy persons; therefore, these results indicated that EBV genome variants probably show no specific tissue distribution.

A reporter system for Epstein-Barr virus (EBV) lytic replication: Anti-EBV activity of the broad anti-herpesviral drug artesunate

Epstein-Barr virus (EBV) is associated with severe human diseases. Therapies with conventional anti-herpesviral drugs are mostly ineffective so that novel drugs are urgently needed. As cell culture-based evaluation systems are required, a GFP (green fluorescent protein) reporter system was generated, which was conceived for an easy quantitation of lytic EBV replication and the analysis of EBV drug sensitivity. A reporter construct was generated on the basis of an EBV plasmid mini-replicon which enabled an episomal maintenance and selection of stably transfected Raji and 293T cell clones. Controlled by the viral lytic origin of DNA replication (oriLyt), this reporter construct could be activated through experimental EBV infection or through chemically stimulated reactivation from EBV latency. Using this system, the sensitivity of EBV to the broad-spectrum anti-herpesviral drug artesunate could be demonstrated: (i) artesunate inhibits EBV in the low micromolar range, (ii) two different strains of EBV are equally artesunate-sensitive, (iii) inhibition is detectable similarly in EBV-infected epithelial cells or lymphocytes, and (iv) the mode of antiviral action is based on a block of viral immediate early protein synthesis. The data demonstrate the usefulness of this reporter system for the quantitation of EBV replication and for determining EBV drug sensitivity.

EBNA1 and LMP1 variants in multiple sclerosis cases and controls

Prior infection with Epstein-Barr virus (EBV) is an established risk factor for multiple sclerosis (MS). Some findings from observational studies, including possible epidemics and differences in prevalence, may be explained if different strains of EBV conferred different MS risk. DNA was extracted from peripheral lymphocytes obtained from 66 MS cases and 66 age- and cohort-matched controls. Nested polymerase chain reaction (PCR) was performed to amplify the N- and C-terminus regions of EBNA1 and the hyper-variable region of the LMP1 gene. For EBNA1, we compared the presence of the prototype B95.8 vs variant sequence and the presence of multiple strains in MS cases and controls. For LMP1, we considered differences in the proportions of mutations between cases and controls. Comparing the proportion of mutant sequence between MS cases and controls in the EBNA1 N-terminal (0/28 vs 1/27) and C-terminal regions (3/40 vs 8/36) revealed no significant differences (P > 0.05). No individual variants in LMP1 were associated with risk of MS (all P > 0.05). Neither EBNA1 nor LMP1 variation was associated with anti-EBNA1 IgG antibody titers. These findings do not support a strong role for variation in EBNA1 N-terminus, EBNA1 C-terminus or LMP1 contributing to MS risk.

Reed-Sternberg cells and "bystander" lymphocytes in lymph nodes affected by Hodgkin's disease are infected with different strains of Epstein-Barr virus.

In most cases of Epstein-Barr virus (EBV)-associated Hodgkin's disease (HD), EBV-positive Reed-Sternberg (RS) cells and rare EBV-positive reservoir lymphocytes coexist in lymph nodes. Here we show that, in two cases of EBV-associated HD, strains infecting RS cells and reservoir lymphocytes of the same patient have different BNLF-1 genes. This suggests that RS cells and reservoir lymphocytes of the same patient are infected by different EBV strains.

Heterogeneity within the Epstein-Barr virus nuclear antigen 2 gene in different strains of Epstein-Barr virus

DNA isolated from biopsies of endemic Burkitt's lymphoma (BL) from New Guinea was analysed for the presence of Epstein-Barr virus (EBV) sequences using the polymerase chain reaction. Primers were designed to amplify sequences within the Epstein-Barr virus nuclear antigen (EBNA) 1 and 2 genes. These analyses detected the EBNA1 sequence in all the biopsies studied. Additional sets of primers directed against the EBNA2 gene were used in order to categorize the EBV strains as A-type or B-type (39% A-type; 50% B-type; 5% A- and B-type; 5% untypeable). These results indicated that DNA sequence heterogeneity within the EBNA2 gene region may exist in different strains of EBV. The extent of DNA sequence heterogeneity among different strains of EBV was determined by sequencing of a region within the EBNA2 gene in a number of different A-type and B-type strains of EBV originating from Africa or New Guinea. The results demonstrated DNA sequence heterogeneity within the EBNA2 gene in different strains of EBV. This heterogeneity was more extensive among A-type strains than B-type strains of EBV.

Amplification and deregulation of MYC following Epstein-Barr virus infection of a human B-cell line.

In Epstein-Barr virus (EBV)-positive Burkitt lymphoma (BL) the role of EBV in the translocation and deregulation of the MYC oncogene remains unknown. By utilizing an EBV-negative BL (BJAB) and several EBV-positive sublines derived from it by in vitro infection, it was possible to show that the presence of the virus was associated with altered expression and copy number of MYC. In the EBV-negative BJAB line, the level of MYC transcripts declined progressively as cells approached the stationary phase of growth. In contrast, in EBV-infected BJAB cells MYC expression remained elevated as cells entered stationary phase. This effect on MYC expression was reversibly linked to the presence of the virus. Furthermore, following EBV infection of BJAB cells by two different strains of EBV, amplification of MYC in association with the appearance of a homogeneously staining region on chromosome 8 at the mapped location of MYC had occurred. These studies suggest that both the deregulation of MYC transcription and the chromosomal rearrangement in the region of the MYC locus in this B-cell line may have occurred as a result of EBV infection.

Effect of 12-O-Tetradecanoylphorbol-13-Acetate (TPA) pn Epstein-Barr Virus (EBV)-Infected Cells

We have examined if 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances soft agar clonability in Epstein-Barr virus (EBV)-infected cells on the basis of interaction between EBV and TPA. When primary human cord blood lymphocytes (HUCLs) were exposed to two different strains of EBV (B95-8 and NPC-KT) and seeded in soft agar medium containing TPA (0.25 ng/ml), it was observed that the number of transformed colonies increased. However, TPA did not enhance the number of colonies in two EBV genome-positive epithelial hybrid cell lines (NPC-KT and D98/HR-1).

Marmoset lymphoblastoid cells transformed by NPC-KT-derived Epstein-Barr virus which possesses transforming and superinfecting characteristics.

New world monkey (cotton-top marmoset; CTM) lymphocytes were transformed by two different strains of Epstein-Barr virus (EBV) derived from human nasopharyngeal epithelial/hybrid cells (NPC-KT and A2L/AH). M-KT cells were a CTM lymphoblastoid cell line which was transformed by EBV derived from NPC-KT cells (NPC-KT EBV). M-BA2L cells were a CTM lymphoblastoid cell line which was transformed by EBV derived from A2L/AH cells (A2L/AH EBV). EBV obtained from M-KT cells, like parental NPC-KT EBV, could transform human cord blood lymphocytes (HCBLs) and superinfect Raji cells. EBV obtained from M-BA2L cells, like parental A2L/AH EBV, could transform HCBLs but could not superinfect Raji cells. We have compared the EBV-DNA associated with M-KT cells to NPC-KT EBV-DNA. The results obtained using a DNA restriction enzyme (Hind III) showed that virus DNA prepared from NPC-KT cells is different from EBV-DNA prepared from CTM lymphocytes transformed by EBV derived from infectious mononucleosis (B95-8), but that virus DNA prepared from M-KT cells is identical with parental NPC-KT EBV. These results indicate the possibility that a single NPC-KT EBV is associated with two biological activities.

Primary EBV Infection of Human Umbilical Cord Lymphocytes and EBV Genome-negative Lymphoblastoid Cell Lines (Bjab and Ramos)

The appearance of Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) and induction of EBV-induced early antigen (EA) in human umbilical cord blood lymphocytes (HUCLs) and two EBV genome-negative Burkitt's lymphoma (BL) lines (BJAB and Ramos) were studied by infection with EBVs prepared from three different cell lines: marmoset cell line (B95-8) derived from infections mononucleosis, BL-derived cell line (P3HR-1) and human epithelial hybrid cell line (NPC-KT) derived from nasopharyngeal carcinoma. B95-8 virus can transform HUCLs but cannot superinfect Raji cells. P3HR-1 virus can transform HUCLs cells but cannot transform HUCLs. NPC-KT virus can transform HUCLs and can superinfect Raji cells. We have examined the time sequence of EBNA appearance and EA induction in HUCLs, BJAB cells and Ramos cells, in order to determine if three different strains of EBV differ in their abilities to infect their cells. We found that all three strains of EBV can induce EBNA in HUCLs, BJAB cells and Ramos cells. On the other hand, we found that P3HR-1 virus and NPC-KT virus can induce EA in BJAB cells and Ramos cells, but B95-8 virus cannot induce EA in their cells.

Neutralization of lymphocyte immortalization by different strains of Epstein-Barr virus with a murine monoclonal antibody

A murine monoclonal antibody was raised against the B95-8 strain of Epstein-Barr virus (EBV), which was isolated from a case of mononucleosis after blood transfusion (Hoffman et al. Proc. Natl. Acad. Sci. U.S.A. 77:2979, 1980). We provide evidence that neutralization of immortalization by this monoclonal antibody is virus specific, since its potency was inversely related to the dose of challenge virus. Furthermore, the monoclonal antibody recognized antigens on viruses grown in human as well as in marmoset cells. We show that this monoclonal antibody neutralized three other transforming strains of EBV originating, respectively, from American patients with mononucleosis and fatal polyclonal lymphoma and from an African child with Burkitt lymphoma. However the antibody did not neutralize or detect antigens by immunofluorescence in the W91 strain of EBV. The hybridoma antibody did neutralize other EBV strains derived from the same Burkitt lymphoma cell line (Nyevu), as was the case with the W91 strain. This monoclonal antibody provides clear evidence of antigenic differences on the surface of EBVs and will ultimately prove useful in defining the antigenic site on EBV which elicits neutralizing antibody.

Antiviral specificities of immunoglobulins secreted by human lymphocytes immortalized in vitro with Epstein-Barr virus

Summary Lymphocytes from a patient (Ma.) with a chronic T-lymphocytic leukaemia were infected with 2 different strains of Epstein-Barr Virus (EBV): B95-8 and M81. Two lymphoblastoid B-cell lines were established; whereas the Ma-M81-cell line secreted a low level of immunoglobulins (Ig) into the culture medium, the Ma-B95-cell line produced unusually large amounts (32 μg/10 6 cells/24 h), mainly IgG. These polyclonal IgG possessed antiviral activities against cytomegalovirus, respiratory syncytial virus, rubella and measles viruses (the patient's serum at the time of lymphocyte immortalization contained IgG antibodies of the same specificities). The Ma-M81-cell line produced complete EBV virions, while in the Ma-B95 cells the replicative cycle of EBV was blocked at the early antigen stage; intracytoplasmic enlarged cisternae were observed in numerous of these latter cells which produced Ig for more than a year.

Epstein-Barr virus (EBV)-lymphoid cell interactions. I. Quantification of EBV particles required for the membrane immunofluorescence assay and the comparative expression of EBV receptors on different human B, T and null cell lines.

We report data on the number of Epstein-Barr virus (EBV) particles required to detect virus binding to target cells (Raji or BJA-B) by means of membrane immunofluorescence (MIF). After determining the optimum conditions for the MIF assay the following aspects of EBV-lymphoid cell interactions were examined: (i) binding of two different strains of EBV to various types of human lymphoid cell lines, (ii) expression of receptors for both EBV and complement on these lines and (iii) induction of EBV-induced nuclear antigen (EBNA) in the different target cells used. The results showed that a minimum of about 2.7 x 10(3) enveloped virus particles/cell were required for an optimum visualization of EBV binding to the target cells tested, and that a lymphoid cell may bear receptors for one prototype strain of EBV but not for the other. A number of cell lines, particularly those of T and null type which express EBV receptors, did not synthesize EBNA, thus indicating that these lines were resistant to EBV infection. Several of these lines, although expressing cell surface EBV receptors, lacked complement receptors.

Epstein–Barr virus genome in infectious mononucleosis

THE seroepidemiological evidence1 linking the Epstein–Barr virus (EBV) causatively with infectious mononucleosis is compelling, but there are many questions concerning the precise nature of the relationship between virus and disease2 and the immunological responses that limit the disease3,4. For example, until recently it remained unproven that the transforming agent excreted from the throat during acute and convalescent infectious mononucleosis is EBV specific. Lymphocytes from the umbilical cord can be transformed into continuous cell lines by exposure to the virus excreted from the throat. New evidence presented here and data published recently show that such transformed lymphocytes contain EBV genetic material as well as a virus-specific nuclear protein5 even though such cells, unlike those isolated from the peripheral blood, do not shed virus. Another question, persistent and crucial, has been whether the EBV originally derived from Burkitt's lymphoma in Uganda and the EBV associated with infectious mononucleosis are identical. We show here that the viral DNA in human lymphocytic lines derived from infectious mononucleosis partially lacks homology to EBV DNA. Such differences imply that an extensively deleted, defective EBV genome is retained in lymphocyte lines established from infectious mononucleosis or that the cell lines harbour different strains of EBV with up to 35 % heterologous DNA sequences.

Characteristics of cell lines derived from human leukocytes transformed by different strains of epstein‐barr virus

Variation of Epstein-Barr virus (EBV) in respect to its effect on the properties of transformed cells was probed. Human umbilical cord leukocytes from six different individuals were transformed in vitro by either B95-8 (B) or QIMR-WIL (Q) strains of EBV and subsequently 12 lymphoblastoid cell lines (six B-derived and six Q-derived lines) were established. The B lines and Q lines were different in the expression of EBV genome i.e. production of virus or viral antigens, and in other properties including growth pattern and immunoglobulin production. The most striking differences between the two groups lay in their capacity to produce infectious virus and in the shape of the cell-clumps. The results suggest that different strains of EBV may induce transformed cells with different characteristics.