Identification of type A and B isolates of Epstein-Barr virus by polymerase chain reaction

A method is described for the identification of type A and type B isolates of Epstein-Barr virus (EBV) by means of the polymerase chain reaction. The use of three pairs of primers specific for genomic sequences coding for the two forms of EBV nuclear antigen (EBNA), 2A and 2B, and for a DNA sequence from the BamZ/BamR region allows the reliable and rapid detection of type A and B viruses in as little as 1000 EBV positive cells.     

A monoclonal antibody that recognizes Epstein-Barr virus nuclear antigen 2 (EBNA 2) amino acids 1–58 does not react with EBNA 2 in native form,consistent with the self-association of EBNA 2 through the amino-terminus

Summary. We have generated a mouse IgG₁ monoclonal antibody (mAb) that recognizes amino acids 1–58 of Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA 2) of type 1 EBV strain B95-8. mAb Y101 also reacted with EBNA 2 of EBV type 2 strains MISP and Jijoye in immunoblots, whereas Jijoye EBNA 2 was not detected by the widely used mAb PE2. mAb Y101, in contrast to mAb PE2, reacted with faster migrated, hypophosphorylated proteins of type 1 EBNA 2 as intensely as slower migrated, hyperphosphorylated ones. mAb Y101 did not react in fixed-cell immunostaining or cell extract immunoprecipitation. The results implicate that the amino-terminal epitope is not exposed in a native form, consistent with the previously reported idea of self-association of EBNA 2 through the amino-terminus. mAb Y101 is the first mAb to the EBNA 2 amino-terminus and will be useful for further analyses of the structure and function of EBNA 2.     

Direct identification by PCR of EBV types and variants in clinical samples

Both Epstein-Barr virus (EBV) type A and type B, and variants of type A, were identified simultaneously by polymerase chain reaction (PCR) amplification of a DNA region coding for a 13 amino acid repeat in the Epstein-Barr virus nuclear antigen (EBNA) 6. Whereas this region varies extensively in type A isolates, no variation was seen in type B isolates. When a repetitive region in the LMP1-coding region was amplified by PCR, it was possible to distinguish individual variants of type B isolates from each other. Forty-two saliva samples from HIV-1-carrying individuals were examined for the presence of type A and type B virus. Both types and multiple variants of each type were found with a much higher frequency than in the saliva samples from healthy individuals. Type A EBV alone was detected in mouthwash samples from 6 infectious mononucleosis (IM) patients. Both type A and B were detected in the peripheral blood B-lymphocytes (PBL) from 1 healthy individual. The same type A variant was demonstrated both in PBL and in the mouthwash sample from another healthy individual. In this study it was shown that a combination of the EBNA 6- and LMP 1-specific PCRs followed by Southern hybridisation can be used to identify both type A and type B virus, as well as to distinguish between multiple variants of the same strain, in saliva and B-cells from both healthy and immunosuppressed individuals. J. Med. Virol. 51:355–363, 1997. © 1997 Wiley-Liss, Inc.     

Induction of nuclear antigen (EBNA) synthesis without transformation in human cord lymphocytes by P3HR-1 strain of Epstein-Barr virus.

Various preparations of P3HR-1 strain of Epstein-Barr virus (EBV) were examined for transforming activity of human cord lymphocytes by a clonal transformation procedure. The B95-8 strain of EBV was used as a positive control. No transformation was evident in all the tests with P3HR-1 virus. However, formation of EBV-associated nuclear antigen (EBNA) without detectable induction of DNA synthesis was demonstrated.     

Presence of Epstein-Barr virus latency type III at the single cell level in post-transplantation lymphoproliferative disorders and AIDS related lymphomas.

AIMS: To investigate the expression pattern of Epstein-Barr virus (EBV) latent genes at the single cell level in post-transplantation lymphoproliferative disorders and acquired immunodefiency syndrome (AIDS) related lymphomas, in relation to cellular morphology. METHODS: Nine post-transplantation lymphoproliferative disorders and three AIDS related lymphomas were subjected to immunohistochemistry using monoclonal antibodies specific for EBV nuclear antigen 1 (EBNA1) (2H4), EBNA2 (PE2 and the new rat anti-EBNA2 monoclonal antibodies 1E6, R3, and 3E9), and LMP1 (CS1-4 and S12). Double staining was performed combining R3 or 3E9 with S12. RESULTS: R3 and 3E9 anti-EBNA2 monoclonal antibodies were more sensitive than PE2, enabling the detection of more EBNA2 positive lymphoma cells. Both in post-transplantation lymphoproliferative disorders and AIDS related lymphomas, different expression patterns were detected at the single cell level. Smaller neoplastic cells were positive for EBNA2 but negative for LMP1. Larger and more blastic neoplastic cells, sometimes resembling Reed-Sternberg cells, were LMP1 positive but EBNA2 negative (EBV latency type II). Morphologically intermediate neoplastic cells coexpressing EBNA2 and LMP1 (EBV latency type III), were detected using R3 and 3E9, and formed a considerable part of the neoplastic population in four of nine post-transplantation lymphoproliferative disorders and two of three AIDS related lymphomas. All samples contained a subpopulation of small tumour cells positive exclusively for Epstein-Barr early RNA and EBNA1. The relation between cellular morphology and EBV expression patterns in this study was less pronounced in AIDS related lymphomas than in post-transplantation lymphoproliferative disorders, because the AIDS related lymphomas were less polymorphic than the post-transplantation lymphoproliferative disorders. CONCLUSIONS: In post-transplantation lymphoproliferative disorders and AIDS related lymphomas, EBV latency type III can be detected by immunohistochemistry in a subpopulation of tumour cells using sensitive monoclonal antibodies R3 and 3E9. Our data suggest that EBV infected tumour cells in these lymphomas undergo gradual changes in the expression of EBV latent genes, and that these changes are associated with changes in cellular morphology.     

Polymorphism analysis of Epstein-Barr virus isolates from patients with cutaneous natural killer/T-cell lymphoproliferative disorders: A possible relation to the endemic occurrence of these diseases in Japan

Certain forms of cutaneous lymphomas in Asia are associated frequently with Epstein-Barr virus (EBV) infection, whereas such cases are less common in western countries. The virus-related peptides, EBV-determined nuclear antigen (EBNA)-2 and the latent membrane protein (LMP)-1, play an essential role in cell transformation. The polymorphisms of these EBV genes may be related to their transforming abilities. In order to clarify the viral subtype that may be involved in the incidence of EBV-associated lymphomas, we analyzed the EBNA-2 and LMP-1 gene polymorphisms and mutations in healthy adults and in patients with EBV-associated cutaneous natural killer(NK)/T-cell lymphoproliferative disorders in Japan. In EBV-related cutaneous lymphoproliferative disorders, EBV subtype 1 was found in all 15 cases, and 1 sample contained a dual infection with subtypes 1 and 2. All EBV isolates from our patients lost a Xho-1 site in exon 1 of the LMP-1 gene, and 7 of 13 cases had a Nco-1 site within the promoter region. All isolates without the LMP-1-Xho-1 site had a 30 bp deletion in the carboxy terminus of the LMP-1 gene, except for the isolate from a patient with angioimmunoblastic lymphadenophathy-like T-cell lymphoma in which a novel Nco-1 site was present in exon 1. Eleven of fourteen throat washings from healthy adults which contained EBV-DNA harbored EBV subtype 1, and the EBNA2 region was not amplified in the other 3 samples. The Xho-1 site was lost in 12 (86%) of 14 isolates and the 30 bp deletion was present in 11 (78%) of 14 isolates from the throat washings. The findings indicate that the predominant EBV isolate from Japanese healthy adults and patients with cutaneous NK/T-cell lymphoproliferative disorders is subtype 1 with a 30 bp deletion and loss of a Xho-1 site in the LMP-1 gene. Since previous data indicated that either subtype 1 or the 30 bp deletion variant possesses high tumorigenic activity, the prevalence of subtype 1 containing these mutations might be responsible for the high incidence of EBV-associated lymphoproliferative disorders in Japan.     

Heterogeneity of Epstein-Barr virus. II. Induction of early antigens (EA) by complementation.

Cells of Epstein-Barr virus (EBV)-negative human B lymphoma lines BJA and Ramos were converted into EBV genome carriers by virus isolates from P3HR-1 and B 95-8 cells (Fresen and zur Hausen, 1976). Cloning of P3HR-1 virus-converted BJA cells resulted in clones with two different Epstein-Barr nuclear antigen (EBNA) patterns: a faint granular EBNA staining and clones with a brilliant EBNA expression (Fresen et al., 1977). The latter always segregated EBNA-negative cells from which one EBNA-negative subclone (B1-28) was isolated. Induction of early antigens (EA) was studied by infecting parental lines (BJA and Ramos), converted lines (BJA-HR1K, BJA-B 95-8, Ramos-HR1K, Ramos-B 95-8), the BJA-HR1K clones A5 (faint granular EBNA expression) and B1-19 (brilliant EBNA expression), the EBNA-negative subclone B1-28, and Raji cells with EBV from P3HR-1 and B 95-8 cells, respectively. The following results were obtained: (1) EA induction by P3HR-1 virus is enhanced on the average 14-fold in EBV genome-harboring cells when compared to genome-negative lines. (2) B 95-8 virus induces EA only in P3HR-1 virus-converted cells and to a small extent also in Raji cells. A significant EA induction occurs in the A5 clone of BJA-HR1K, whereas the brilliantly EBNA-expressing B1-19 clone is not induced. B 95-8 virus-converted cells cannot be induced by B 95-8 virus. (3) EA induction following infection of EBV genome-carrying cells is directly proportional to the dilution of the infecting virus. In EBV genome-free cells, EA induction is reduced by the square of the dilution factor. These results imply that resident genomes complement superinfecting genomes in EA induction by EBV and that two different populations of genomes (present in P3HR-1 virus isolates) are required for EA induction following infection of B lymphoblasts.     

Synthetic peptides deduced from the amino acid sequence of Epstein-Barr virus nuclear antigen 6 (EBNA 6): Antigenic properties,production of monoreactive reagents,and analysis of antibody responses in man

Studies on the antibody responses to various Epstein-Barr virus (EBV) antigens have been instrumental in the understanding of the seroepidemiology and diagnosis of this viral infection and the subsequent carrier state. While antibodies to the viral capsid antigen (VCA), early antigen (EA), and nuclear antigens 1 and 2 (EBNA 1 and 2) have been well characterized, the antibody response to the other nuclear antigens is not well understood. EBNA 6 is expressed by lymphoblasts during acute EBV infection and may be an important antigen for diagnosis and evaluation of the immune response. In order to analyze the antibody response to EBNA 6, ten peptides (20–21 amino acids), deduced from the EBNA 6 coding region, were synthesized and evaluated for antigenicity by ELISA. One peptide (p-63; PA-PQAPYQGYQEPPAPQAPY) derived from the amino acid repeats showed the highest specific reactivity with human sera. This peptide was evaluated further for detection of human EBNA 6-reactive antibodies. Forty-two of forty-nine (86%) EBV-seropositive healthy donors had p-63- specific IgG reactivity, while none of 50 EBV seronegative patients reacted with the p-63 peptide. Twenty-two of fifty-one (43%) patients with ongoing primary EBV infection had detectable p-63-specific IgG. Serum samples drawn sequentially from patients during and after primary EBV infection revealed an increase in p-63-reactive IgG over time. A similar pattern was found for reactivity with an EBNA I-specific peptide (p-107), in contrast to the EBNA 2 (polyproline) response, which decreased over time. Some EBV seropositive individuals who had no detectable IgG against peptide p-63 did have antibodies against the native EBNA 6 by anticomplement immunofluorescence to EBNA 6 transfected cells. Rabbit antiserum raised against p-63 reacted specifically with native EBNA 6 by an immunofluorescence assay and by immunoblotting, indicating the EBNA 6-specific antigenicity of the peptide. Thus, the peptide p-63 derived from the amino acid repeats of the EBNA 6 coding region constitutes a predominant, although not exclusive, epitope in the EBNA 6 antibody response. © 1995 Wiley-Liss, Inc.     

Blocking of acid-fixed nuclear binding of Epstein-Barr virus nuclear antigen (EBNA) by different DNA species.

Treatment of Epstein-Barr virus-determined nuclear antigen (EBNA) with DNA resulted in blocking of its ability to convert acid-fixed EBNA-negative cell nuclei to an EBNA-positive form. Epstein-Barr virus (EBV) DNA, herpes simplex virus type 2 (HSV-2) DNA and DNA isolated from three lymphoblastoid cell lines differed in their potency to block this reaction. EBV DNA was found to be about three times more effective than cellular DNAs in abolishing the ability of DNA-cellulose-purified EBNA to convert acid-fixed nuclei to the EBNA-positive form; the effect of HSV-2 DNA was of intermediate character. No difference was found between the blocking potency of DNAs isolated from EBV-genome-negative Ramos cells and EBV-genome-positive Raji and P3HR-1 cells.     

Epstein-Barr virus nuclear antigen 1 (EBNA1) induced cytotoxicity in epithelial cells is associated with EBNA1 degradation and processing

Epstein-Barr virus nuclear antigen 1 (EBNA1) has a central role in the maintenance and segregation of the Epstein-Barr virus (EBV) episome and by virtue of a glycine-alanine repeat domain is prevented from being endogenously processed for recognition by HLA class I restricted cytotoxic T lymphocytes (CTLs). We found that EBNA1 expression resulted in growth inhibition and a G2/M arrest in human squamous epithelial cell lines (SCC12F, SVK) but not epithelial cell lines of glandular origin (Hela, Ad/AH). The cytotoxicity of EBNA1 was associated with EBNA1 degradation and both these effects were blocked in SCC12F cells expressing either the anti-apoptotic bcl-2 protein or the EBV homolog of bcl-2, BHRF1. The endogenous degradation of EBNA1 in SVK epithelial cells was associated with specific CTL recognition, an effect not evident in EBNA1-expressing Hela cells. Consistent with the inability of SVK cells to tolerate EBNA1 expression, studies with a recombinant EBV demonstrated that SVK cells are unable to maintain stable virus infection, whereas Hela cells are able to efficiently establish latent EBV infection. These data have important implications for both the cellular requirements necessary to sustain a stable EBV infection and for the possible role of CTL responses in controlling EBV infection of epithelial cells.     

Characteristics of viral protein expression by Epstein-Barr virus-infected B cells in peripheral blood of patients with infectious mononucleosis.

The frequency of Epstein-Barr virus (EBV) antigen-positive B cells in the peripheral blood of patients with infectious mononucleosis compared with that for latently EBV-infected individuals was examined by immunocytochemistry. B cells positive for Epstein-Barr nuclear antigen (EBNA) 1, EBNA2, and latent membrane protein were frequently found in all peripheral B lymphocyte preparations from 25 patients suffering for 3 to 28 days from infectious mononucleosis by using monoclonal antibodies and the alkaline phosphatase anti-alkaline technique. There was a significant decrease in the number of positive B cells during the course of disease. EBNA1-positive B cells were detected in 0.01 to 2.5% of total B cells (median, 0.8%), EBNA2-positive B cells were detected in 0.01 to 4.5% of total B cells (median, 0.9%), and latent membrane protein-positive B cells were detected in 0.01 to 1.8% of total B cells (median, 0.5%), depending on the duration of clinical signs. In contrast, we did not find any EBNA1- or EBNA2-positive B cells in 2 x 10(6) peripheral blood B lymphocytes of 10 latently EBV-infected individuals, whereas aliquots of the same cell preparations were EBV DNA positive by a PCR assay. Therefore, it appears to be possible to detect infectious mononucleosis by immunocytochemical determination of latent EBV products, which might be of relevance for the diagnosis of EBV reactivations in immunosuppressed patients.     

An EBNA-negative, EBV-genome-positive human lymphoblast cell line in which superinfecting EBV DNA is not maintained

A human B-lymphoid cell line, designated TG8, which does not express detectable levels of the EBV (Epstein-Barr virus)-associated nuclear antigen (EBNA), yet carries an average of one to two plasmid copies of the P3HR-1 EBV genome has been identified. TG8 can be superinfected by B95-8 EBV, resulting in up to 60-70% of the population becoming EBNA-positive and 20-30% of the incoming EBV genomes becoming circular by 48 hr postinfection. Neither EBNA expression nor the superinfecting viral DNA is maintained in the population. It is concluded that (1) superinfection of this EBV-genome-positive lymphoblast cell line leads to detectable EBNA expression and circularization of the incoming viral genome and (2) the incoming viral genome and detectable EBNA expression are selectively lost, whereas the endogenous viral plasmid DNA is maintained.     

Epstein-Barr virus nuclear antigen 1 evades direct immune recognition by CD4+ T helper cells

The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein regularly expressed in EBV-associated malignancies. Immune recognition of EBNA1 by CD8+ T cells is prevented by an internal glycine-alanine repeat (GAr) which blocks proteasomal degradation. To test whether EBV-infected cells could be recognized by T helper cells, human CD4+ T cell clones specific for EBNA1 were isolated from latently EBV-infected individuals. These T cells, however, failed to recognize EBV-positive target cells. To investigate whether endogenous presentation of EBNA1 epitopes on MHC class II was prevented by the GAr domain, a mutant EBV strain with an EBNA1 lacking the GAr (EBNA1DeltaGA) was generated and used to establish an Epstein-Barr virus-immortalized lymphoblastoid B cell line (LCL). The EBNA1DeltaGA LCL were not recognized by the EBNA1-specific T cell clones either, indicating that the GAr domain does not mediate this effect. Immune recognition could be restored by overexpression of EBNA1, for which at least 60-fold higher levels of both EBNA1 or EBNA1DeltaGAr protein were required. These results demonstrate that EBNA1 evades direct recognition by CD4+ T helper cells, since its steady state level is below the threshold required for efficient presentation on MHC class II. These findings have important implications for the design of immunotherapeutic approaches to target EBV-positive malignancies.     

Characterization of an Epstein-Barr virus nuclear antigen 2 variant (EBNA 2B) by specific sera

The Jijoye Epstein-Barr virus (EBV) strain is characterized by a substitution of 1.8 kb in the C-terminal part of EBNA 2 gene compared to B95-8 or M-ABA virus. Protein immunoblot analysis using human sera against EBNA 2 indicated that an immunological variant to the EBNA 2 of B95-8 (type A) is encoded by the Jijoye virus (type B). In order to generate a specific EBNA 2B antiserum the NaeI/NsiI DNA fragment of the Jijoye virus containing 237 bp of the C-terminus from the EBNA 2B gene was cloned in an E. coli expression vector (pME3). The resulting fusion protein contained 79 C-terminal amino acids of the viral protein and a 37,000 Da part of the bacterial anthranilate synthase. Rabbit antisera generated against this fusion protein reacted specifically with two proteins of 73,000 and 77,000 Da from Jijoye cells and three other cell lines carrying type B virus, while no proteins could be identified in the type B cell line BL 29. In addition, using these sera directed against the pME3 fusion protein, no reaction could be observed with the EBNA 2A protein from the B95-8 and several other cell lines containing type A virus.     

Analysis of the transformation of human lymphocytes by Epstein-Barr virus III

T-cell depleted human cord blood lymphocytes were exposed to P3HR-1 strain of Epstein-Barr virus (EBV) and simultaneous observations of immunofluorescence, cellular morphology, and autoradiography were carried out in individual cells. Soon after infection, nuclear antigen (EBNA) synthesis, blastogenesis, and DNA synthesis occurred, as was previously observed in B95-8 strain EBV infection. Although mitosis followed with characteristic cell aggregate formation, the cell proliferation was temporary and death followed in about 2 weeks. The synthesis of the early antigens (EA) and the viral capsid antigen (VCA) were not significant. These findings seem to indicate that the strain P3HR-1 EBV is capable of inducing early events of transformation in primary human B-lymphocytes, but the cells infected in this way have a short life span.     

Correlation of the expression of Epstein-Barr virus latent membrane protein and in situ hybridization with biotinylated BamHI-W probes in Hodgkin''s disease.

The detection of Epstein-Barr virus (EBV) nucleic acids by in situ hybridization (ISH) with biotinylated BamHI-W probes was correlated with the expressions of EBV latent membrane protein (LMP) and EB nuclear antigen 2 (EBNA2), in 107 cases of Hodgkin's disease (HD) of different immunomorphologic subtypes. Epstein-Barr virus nucleic acids were present and restricted to the pathogenic cells in 4 of 40 (10%) cases of nodular sclerosis (NS) and 33 of 55 (60%) cases of mixed cellularity (MC), but were undetectable in other subtypes. Of the 37 cases positive for EBV nucleic acids, 35 (95%) showed the expression of LMP. Epstein-Barr virus nucleic acids and LMP were restricted to Reed-Sternberg cells and variants. Only 1 case (MC) showed LMP expression in the absence of EBV detection. The correlation was strengthened by the finding of LMP expression at first diagnosis in 6/7 EBV positive cases at relapse (14-126 months) (5/5 EBV negative cases at relapse were LMP negative at first diagnosis). EBNA2 was absent in all 13 (NS, 2; MC, 11) EBV+ and LMP+ cases tested. Both LMP and EBNA2 were expressed in control EBV-positive tissues and cell lines. EBV serology in MC HD was indicative of latent EBV infection, but neither serology nor clinical parameters correlated with the presence or the absence of EBV, over a short-term follow-up (median, 20 months). The findings, although not proving EBV as the etiologic agent of HD, suggest that: 1) LMP expression alone may be adequate for identifying EBV-associated HD, 2) the MC subtype has a stronger relation with EBV presence, and 3) the regulation of EBV genes in HD is different from other EBV-associated disorders. The clinical implications still remain to be discovered.