EPSTEIN–BARR VIRUS (EBV) INFECTION IN INFECTIOUS MONONUCLEOSIS: VIRUS LATENCY,REPLICATION AND PHENOTYPE OF EBV-INFECTED CELLS

Primary Epstein–Barr virus (EBV) infection may manifest itself as a benign lymphoproliferative disorder, infectious mononucleosis (IM). EBV infection has been characterized in lymphoreticular tissues from nine patients with IM using the abundantly expressed EBV-encoded nuclear RNAs (EBERs) as a marker of latent infection. Expression of the virus-encoded nuclear antigen (EBNA) 2 and of the latent membrane protein (LMP) 1 was seen in variable proportions of cells in all cases. Double labelling revealed heterogeneous expression patterns of these proteins. Thus, in addition to cells revealing phenotypes consistent with latencies I (EBNA2⁻/LMP1⁻) and III (EBNA2⁺/LMP1⁺), cells displaying a latency II pattern (EBNA2⁻/LMP1⁺) were observed. Cells expressing EBNA2 but not LMP1 were also detected; whilst this may represent a transitory phenomenon, the exact significance of this observation is at present uncertain. EBER-specific in situ hybridization in conjunction with immunohistochemistry revealed expression of the EBERs mainly in B-lymphocytes, many of which showed features of plasma cell differentiation. By contrast, convincing evidence of latent EBV infection was not found in T-cells, epithelial or endothelial cells. Double-labelling immunohistochemistry revealed expression of the replication-associated BZLF1 protein in small lymphoid cells, often showing plasmacytoid differentiation. There was no unambiguous expression of this protein in other cell types. These results suggest that B-cells are the primary target of EBV infection and that plasma cells may be a source of infectious virus found in the saliva of IM patients. © 1997 John Wiley & Sons, Ltd.     

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.     

Elevated IgA antibodies to Epstein-Barr virus in children with chronic active Epstein-Barr virus infection

Anti-Epstein-Barr virus (EBV) antibodies were tested in 11 children with chronic active EBV infection. Anti-virus capsid antigen (VCA)-IgG antibody titers ranged from 1:640 to 1:10,240. Anti-VCA-IgM antibody was consistently positive in 5 of the 11 patients; anti-VCA-IgA antibody was consistently positive in 6 of the 10 patients; anti-early antigen (EA)-IgG antibody was consistently positive in 10 of the 11 patients and anti-EA-IgA antibody was consistently positive in 4 out of the 7 patients. Anti-EBV nuclear antigen (EBNA) antibody was not detected in two patients. Consistently positive anti-VCA-IgA- and anti-EA-IgA- antibody may be a characteristic feature of abnormal antibody responses in severe chronic active EBV-infection in childhood.     

Antibody responses to two Epstein-Barr virus nuclear antigens defined by gene transfer

By transfecting small fragments of Epstein-Barr virus (EBV) DNA into cells, we defined two nuclear antigens, termed M and K, and examined serum from 258 subjects for antibodies against these antigens. We hoped to learn whether such single-antigen systems would clarify the association of EBV with various diseases. Although reactivity to M antigen was found in only 14 per cent of healthy EBV-seropositive subjects, 90 per cent of Chinese and North African patients with nasopharyngeal carcinoma had antibody to M. Nearly all persons (96 per cent) who were EBV seropositive, as judged by their serologic reaction to a nuclear antigen encoded by the complete virus (EBNA), had a reaction to K antigen. However, serum samples from three patients with chronic active EBV infection did not react to K, even though the serum contained anti-M titers above 1:1000. Lymphoid cells from one such patient carried a normal gene for K and made K protein of correct size. Therefore, in this patient the absence of antibody to K had not resulted from a viral mutation that destroyed the K protein. These serologic studies show that some patients with chronic active EBV infection have an abnormal immune response to a specific viral gene product.     

Epstein-Barr virus latent and replicative gene expression in oral hairy leukoplakia

Oral hairy leukoplakia is an epithelial lesion of the tongue associated with productive infection by Epstein-Barr virus (EBV). However, no data concerning the pattern of EBV latent gene expression have been reported, and it remains unresolved whether true latent infection occurs in basal cell layers of oral hairy leukoplakia. We have studied six cases of oral hairy leukoplakia using monoclonal antibody immunohistology for EBV latent--EB nuclear antigen (EBNA) 1, EBNA 2 and latent membrane protein 1 (LMP 1); immediate-early (BZLF1); and replicative (EA, VCA, MA) proteins, and for the EBV-receptor (CD21 antigen). EBV DNA was demonstrated by nucleic acid in situ hybridization. Mid- to upper-zone keratinocytes contained EBV DNA and co-expressed EBNA 1, EBNA 2 (5 of 6 cases), LMP 1, BZLF1 protein, EA, VCA and MA. No EBV genome or gene expression could be demonstrated in basal or parabasal cells. Spinous keratinocytes were labelled by anti-CD21 antibodies HB5 and B2, but did not express the EBV-receptor as defined by reactivity with OKB7. The co-expression of latent and replicative infection-associated antigens is striking, indicating possible functional roles for latent proteins during the productive cycle. Our results suggest that oral hairy leukoplakia is caused by repeated direct infection of upper epithelial cells with virus from saliva or adjacent replicatively infected cells, rather than by a latent EBV infection of basal epithelial cells with a differentiation-dependent switch to productive infection as previously proposed.     

IN SITU DETECTION OF THE EPSTEIN–BARR VIRUS-ENCODED NUCLEAR ANTIGEN 1 IN ORAL HAIRY LEUKOPLAKIA AND VIRUS-ASSOCIATED CARCINOMAS

A new monoclonal antibody has been used to examine immunohistochemically the expression of the Epstein–Barr virus (EBV)-encoded nuclear antigen (EBNA) 1 in virus-associated epithelial lesions. EBNA1 was detected in the tumour cell nuclei of 10/13 undifferentiated nasopharyngeal carcinomas and of 10/10 EBV-associated gastric carcinomas. EBNA1 was also detected in 13 of 16 oral hairy leukoplakia (HL) samples, where its expression was confined to nuclei in the upper epithelial cell layers whilst basal epithelial cells were negative. This observation is in agreement with previous studies demonstrating the absence of latent EBV infection in the basal cell compartment of HL and suggests an essential role for EBNA1, not only in latent EBV infection but also in virus replication.     

A human monoclonal autoantibody isolated from a patient with infectious mononucleosis reactive with both self antigens and Epstein-Barr virus nuclear antigen (EBNA)

In order to investigate the mechanism(s) by which Epstein-Barr virus (EBV) induces the outcome of autoantibodies during infectious mononucleosis (IM), a human IgM (k) monoclonal antibody to cytoskeletal filaments of epithelial cells has been prepared by EBV transformation of peripheral blood B lymphocytes obtained from a patient with IM. The antibody was also found to react with smooth muscle of frozen sections of human stomach tissue by immunofluorescence, and with the Epstein-Barr nuclear antigen (EBNA) by an enzyme-linked immunosorbent assay. These findings demonstrate at the clonal level the epitope homology between host's cell antigens and EBV-encoded nuclear antigen, which might have relevance in EBV-induced autoimmunity.     

Epstein-Barr virus and multiple sclerosis: interaction with HLA

Epstein-Barr virus (EBV) infection, history of infectious mononucleosis (IM) and HLA-A and DRB1 have all been proposed as risk factors for multiple sclerosis (MS). Our aim was to analyse possible interactions between antibodies against Epstein-Barr virus nuclear antigen 1 (EBNA1) or EBNA1 fragments, presence of DRB1*15 and absence of A*02. The study population includes newly diagnosed cases and matched controls. Interaction on the additive scale was calculated using attributable proportion due to interaction (AP), which is the proportion of the incidence among individuals exposed to two interacting factors that is attributable to the interaction per se. IM showed association with MS, odds ratio (OR)=1.89 (1.45-2.48% confidence interval (CI)), as did raised EBNA1 IgG OR=1.74 (1.38-2.18 95%CI). All EBNA1 fragment IgGs were associated with MS risk. However, EBNA1 fragment 385-420 IgG levels were more strongly associated to MS than total EBNA1 IgG, OR=3.60 (2.75-4.72 95%CI), and also interacted with both DRB1*15 and absence of A*02, AP 0.60 (0.45-0.76 95%CI) and AP 0.39 (0.18-0.61 95%CI), respectively. The observed interaction between HLA class I and II genotype and reactivity to EBV-related epitopes suggest that the mechanism through which HLA genes influence the risk of MS may, at least in part, involve the immune control of EBV infection.     

Evaluation of 12 commercial tests for detection of Epstein-Barr virus-specific and heterophile antibodies

Ten microbiological departments in Norway have participated in a multicenter evaluation of the following commercial tests for detection of Epstein-Barr virus (EBV)-specific and heterophile antibodies: CAPTIA Select viral capsid antigen (VCA)-M/G/EBNA (Centocor Inc.), Enzygnost anti-EBV/immunoglobulin M (IgM) and IgG (Dade Behring), Vironostika EBV VCA IgM/IgG/EBNA enzyme-linked immunosorbent assay (ELISA) (Organon Teknika), SEROFLUOR immunofluorescence assay and EBV Combi-Test (Institute Virion Ltd.), anti-EBV recombinant IgM- and IgG-early antigen/EBNA IgG ELISA (Biotest Diagnostics), EBV IgM/IgG/EBNA ELISA (Gull Laboratories), Paul-Bunnell-Davidsohn test (Sanofi Diagnostics Pasteur), Monosticon Dri-Dot (Organon Teknika), Avitex-IM (Omega Diagnostics Ltd.), Alexon Serascan infectious mononucleosis test (Alexon Biomedical Inc. ), Clearview IM (Unipath Ltd.), and Cards+/-OS Mono (Pacific Biotech, Inc.). The test panel included sera from patients with primary EBV infection, immunocompromised patients with recent cytomegalovirus infection, healthy persons (blood donors), and EBV-seronegative persons. Among the tests for EBV-specific antibodies the sensitivity was good, with only small differences between the different assays. However, there was a greater variation in specificity, which varied between 100% (Enzygnost) and 86% (Biotest). Tests for detection of heterophile antibodies based on purified or selected antigen (Avitex, Alexon, Clearview IM, and Cards+/-OS Mono) were more sensitive than the Paul-Bunnell-Davidsohn and Monosticon tests.     

Evaluation of 12 Commercial Tests for Detection of Epstein-Barr Virus-Specific and Heterophile Antibodies

Ten microbiological departments in Norway have participated in a multicenter evaluation of the following commercial tests for detection of Epstein-Barr virus (EBV)-specific and heterophile antibodies: CAPTIA Select viral capsid antigen (VCA)-M/G/EBNA (Centocor Inc.), Enzygnost anti-EBV/immunoglobulin M (IgM) and IgG (Dade Behring), Vironostika EBV VCA IgM/IgG/EBNA enzyme-linked immunosorbent assay (ELISA) (Organon Teknika), SEROFLUOR immunofluorescence assay and EBV Combi-Test (Institute Virion Ltd.), anti-EBV recombinant IgM- and IgG-early antigen/EBNA IgG ELISA (Biotest Diagnostics), EBV IgM/IgG/EBNA ELISA (Gull Laboratories), Paul-Bunnell-Davidsohn test (Sanofi Diagnostics Pasteur), Monosticon Dri-Dot (Organon Teknika), Avitex-IM (Omega Diagnostics Ltd.), Alexon Serascan infectious mononucleosis test (Alexon Biomedical Inc.), Clearview IM (Unipath Ltd.), and Cards±OS Mono (Pacific Biotech, Inc.). The test panel included sera from patients with primary EBV infection, immunocompromised patients with recent cytomegalovirus infection, healthy persons (blood donors), and EBV-seronegative persons. Among the tests for EBV-specific antibodies the sensitivity was good, with only small differences between the different assays. However, there was a greater variation in specificity, which varied between 100% (Enzygnost) and 86% (Biotest). Tests for detection of heterophile antibodies based on purified or selected antigen (Avitex, Alexon, Clearview IM, and Cards±OS Mono) were more sensitive than the Paul-Bunnell-Davidsohn and Monosticon tests.     

The seroepidemiology of infection due to Epstein-Barr virus in southern India

We investigated the seroepidemiology of infection due to Epstein-Barr virus (EBV) in 181 south Indian subjects aged 0-25 years using the indirect immunofluorescence method to titrate antibodies to viral capsid antigen (VCA), nuclear antigen (EBNA), and early antigen (EA). The age-specific prevalence of IgG antibodies to VCA rose rapidly to 90% by the age of 5 years. The prevalence of VCA-specific IgM and the geometric mean titre of VCA-specific IgG antibodies were highest between the ages of 6 months and 2 years, the median age of primary infection being 1.4 years. Thus primary EBV infection occurs early in life. EA antibody prevalence was highest (55%) in the third year of life and remained between 30% and 40% thereafter. This pattern of EA antibody prevalence suggests that the latent EBV infection that persists lifelong after primary infection may be reactivated in many individuals. EBNA antibody prevalence was low until the age of 2 years but rose to 80% in the fourth year. Geometric mean titres of antibodies to EA and EBNA were low and stable at all ages. These results are similar to data from areas where EBV-associated Burkitt's lymphoma is endemic and indicate a high EBV infection load early in life.     

Epstein-Barr virus (EBV) antibodies in children with non-Hodgkin's lymphomas

Antibody titres against Epstein-Barr virus (EBV) antigens in children suffering from non-Hodgkin's lymphoma (NHL) were determined. IgG antibody titres against the viral capsid antigen (VCA) and early antigen (EA) exceeded those found in healthy control subjects. On the other hand, antibody titres against EBV-determined nuclear antigen (EBNA complex) were generally lower than in the control group. The most striking phenomenon observed in the patient group was the frequent activation of latent virus infection as revealed by the periodical appearance of anti-EA and IgM class anti-VCA antibodies. Antibody titres against EBV antigens were generally lower among patients with progressing disease than in those with a more favourable course of the illness. The closest relation to EBV based on serological findings, was detected in lymphoblastic lymphomas of Burkitt-type histology, poorly differentiated lymphocytic lymphomas, and in lymphomas localized in the abdomen. The question whether EBV might be involved in a certain proportion of the cases examined is discussed and further approaches to elucidate this problem are suggested.     

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.     

The antibody 2B4 directed against the Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) detects MAGE-4: implications for studies on the EBV association of human cancers

We have previously developed two monoclonal antibodies against the Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA1), designated 1H4 and 2B4. Both detect EBNA1 by in situ staining in established EBV-positive tumours, e.g. Hodgkin's lymphoma and nasopharyngeal carcinoma. An association of EBV with other tumours, notably breast carcinomas, has been reported but remains controversial. Using the antibody 2B4, a nuclear protein has been detected in breast carcinomas that were EBV-negative by other methods, suggesting cross-reactivity with a cellular protein. Furthermore, an association of EBV with various other carcinomas has been reported on the basis of 2B4 immunohistochemistry. Here we show that 2B4 also binds to MAGE-4, a cancer testis antigen expressed in a variety of tumour cells, including breast carcinoma, seminoma and EBV-negative cases of Hodgkin's lymphoma. We conclude that the 2B4 antibody is not suitable for the detection of EBV infection but that additional techniques, particularly in situ hybridization for the detection of the EBV-encoded RNAs (EBERs), should be employed to confirm the presence of EBV. Our results add to the evidence indicating that breast cancer is not an EBV-associated disease.     

Evaluation of commercial EBV RecombLine assay for diagnosis of nasopharyngeal carcinoma

BACKGROUND: In recent years a number of Epstein-Barr virus (EBV) proteins were defined as being immunodominant for either IgM, IgG or IgA immune responses, yielding promising markers for diagnostic serology. Specific reactivity patterns to these proteins have been described for infectious mononucleosis (IM), nasopharyngeal carcinoma (NPC), various types of lymphoma, and healthy EBV carriers. OBJECTIVES: To compare the NPC-related diagnostic value of EBV RecombLine test (Mikrogen, Germany) with a standardized immunoblot assay [Fachiroh J, Schouten T, Hariwiyanto B, Paramita DK, Harijadi A, Haryana SM, et al. Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects. J Infect Dis 2004;190:53-62] and to define the diagnostic value of individual EBV marker proteins in a population with high incidence of NPC. RESULT: Sera from Indonesian NPC patients taken at primary diagnosis (n=108) were analyzed for IgG and IgA reactivity and compared with regional healthy blood donors (n=62), non-NPC patient controls (n=10) and IM patients (n=10). Most NPC patients and controls showed strong IgG reactivity to VCA-p18, -p23, and EBNA1, limiting their diagnostic use. Few (<20%) healthy donors and patient controls showed IgG reactivity to EA proteins p47/54 and p138, yielding combined sensitivity/specificity and PPV/NPV values of 92.6%/98.3% and 99.0%/88.1%, for diagnosing NPC. NPC sera showed significantly more EBV reactive IgA antibody (>80% positive) than controls (<10% positive), although being less broadly reactive and significantly less strong compared to IgG. For IgA best results were observed for RecombLine EBNA1 with sensitivity/specificity and PPV/NPV values of 92%/89% and 93.4%/85.9%, respectively. CONCLUSION: In high incidence NPC regions with low incidence IM yet high prevalence of EBV infection, both RecombLine IgG and IgA tests provide a useful alternative to the more complex cell-extract based immunoblot assay as confirmation test for NPC diagnosis in particular when using EA and EBNA1 as discriminators in IgG and IgA testing, respectively.     

Syncytium-forming virus of common marmosets (Callithrix jacchus jacchus).

This communication describes the isolation and characterization of a new syncytium-forming virus of common marmosets (Callithrix jacchus jacchus). The virus, isolated from skin explants and peripheral leukocytes of healthy animals, induced syncytia and subsequent cytolysis of several human, simian, and rodent fibroblastic cultures and induced a carrier state in mixed fibroblastic-epithelial or epithelial cell lines. Cytoplasmic and nuclear viral antigen was demonstrated in infected cells by indirect immunofluorescence tests using serum obtained from persistently infected common marmosets. Abundant virus particles were detected within cisternae of endoplasmic reticulum of lytically infected cells by electron microscopy. The virus incorporated [3H]uridine, banded at a density of 1.14 to 1.16 g/cm3 in sucrose, and possessed ribonucleic acid-dependent deoxyribonucleic acid polymerase. No antigenic cross-reactivity was detected between the marmoset virus and simian foamy virus serotypes 1 to 8 in neutralization and immunofluorescence assays. A seroepidemiological survey of a marmoset colony revealed that 53.5% of common marmosets contained antibodies against the virus, whereas other species of marmosets maintained in the same colony remained free of antibodies.     

EBNA1 sequences in Argentinean pediatric acute and latent Epstein–Barr virus infection reflect circulation of novel South American variants

Epstein–Barr virus (EBV) is related to the development of lymphomas and is also the etiological agent for infectious mononucleosis (IM). Sequence variation of the EBNA1 gene, consistently expressed in all EBV‐positive cells, has been widely studied. Based on the amino acid at codon 487 five major EBNA1 variants have been described, two closely related prototypic variants (P‐ala and P‐thr) and three variant sequences (V‐leu, V‐val, and V‐pro). Sub‐variants were then further classified based on mutations other than the originally described. While several studies proposed associations with tumors and/or anatomical compartments, others argued in favor of a geographical distribution of these variants. In the present study, EBNA1 variants in 11 pediatric patients with IM and 19 pediatric EBV lymphomas from Argentina were compared as representatives of benign and malignant infection in children, respectively. A 3‐month follow‐up study of EBNA1 variants in peripheral blood cells and in oral secretions of patients with IM was performed. A new V‐ala variant which includes five V‐ala sub‐variants and three new V‐leu sub‐variants was described. These data favor the geographical association hypothesis since no evidence for a preferential compartment distribution of EBNA1 variants and sub‐variants was found. This is the first study to characterize EBNA1 variants in pediatric patients with infection mononucleosis worldwide. J. Med. Virol. 82:1730–1738, 2010. © 2010 Wiley‐Liss, Inc.     

Distinct patterns of viral antigen expression in Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus coinfected body-cavity-based lymphoma cell lines: potential switches in latent gene expression due to coinfection.

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8 (HHV-8), are human gammaherpesviruses associated with numerous lymphomas and proliferative diseases in humans. We were interested in the protein expression patterns of specific latent and lytic proteins from the EBV genome in two body-cavity-based lymphoma cell lines, BC-1 and BC-2, which are coinfected with EBV and KSHV. BC-1 and BC-2 were analyzed using specific antibodies to latent proteins known to be essential for EBV immortalization of human primary B-lymphocytes in vitro and lytic antigens important for EBV replication and production of viral progeny. The coinfected cell lines are compared with two singly infected KSHV cell lines to determine whether antibodies against EBV-specific proteins cross-reacted against KSHV antigens. All the KSHV-infected cell lines express the KSHV-specific latency-associated nuclear antigen (LANA) with a specific pattern in the nucleus. This staining was distinct from that seen for EBNA1 in the EBV coinfected lines BC-1 and BC-2 staining the nucleus as a diffused pattern throughout the nucleus with denser staining in some regions. The coinfected cell lines all express EBNA1 and LMP1 at lower levels compared with singly infected EBV lymphoblastoid cell lines (LCLs). However, the essential latent antigens EBNA2, EBNA3A, and EBNA3C are not expressed in BC-1 and BC-2. This indicates potential regulation of EBV latent gene expression by KSHV-encoded viral or KSHV-induced cellular gene products. Additionally, lytic gene expression analysis demonstrated that BZLF1 and BMRF1 are expressed along with other early antigens (EA-D). A specific protein is detected in a singly infected KSHV cell line with cross-reactivity to antibodies that detected the EA-D complex. Moreover, in all the cell lines infected with EBV, KSHV, or EBV and KSHV, human serum with antibodies against KSHV antigens recognizes specific viral antigens approximately 110 and 41-42 kDa, suggesting that human antibodies against KSHV-specific antigens can cross-react with similar EBV antigens. Therefore these data suggest that the EBV pattern of gene expression in the coinfected cell lines is a type II pattern of latency also seen in other human tumors including nasopharyngeal carcinoma and Hodgkin's lymphoma. This distinct pattern of latent and lytic gene expression in these cell lines may provide clues as to the selection for coinfection in these body cavity based lymphomas in immunocompromised hosts.     

Effects of DNA synthesis inhibitors on early antigen expression following primary infection or superinfection by Epstein-Barr virus

Summary Seven lymphoid cell lines previously characterized with respect to their resident Epstein-Barr virus (EBV) genome content were infected or superinfected with concentrated EBV from supernatant of the P3 HR-1 cell line. Immunofluorescence assays were conducted on smears 48 hours after infection, using human sera containing antibodies to EBV early antigen (EA). Two EBV nuclear antigen (EBNA) negative cell lines containing no detectable resident EBV DNA and five EBNA positive cell lines containing EBV genomes were tested. The cell lines did not spontaneously express EBV EA (i.e., they were non-producers). All cell lines responded to infection or superinfection with EBV by expressing EA. Treatment of the cell lines with arabinosylcytosine (Ara-C) 10 µg/ml, at the time of infection resulted in significant decreases in the number of cells expressing detectable EA after drug treatment in all cell lines (72±5 percent inhibition of EA expression). Experiments were also conducted with hydroxyurea (HU) and phosphonoacetic acid (PAA). It was found that treatment with HU (100 µg/ml) inhibited EA production in cell lines containing EBV genome copies by 81 percent as compared to the superinfected cultures receiving no drug. In primary infection of EBNA negative cell lines, HU had minimal effects. PAA (100 µg/ml), on the other hand, had very little effect on EA expression following superinfection of cell lines harboring the EBV genome, but reduced the EA expression after primary infection of EBNA negative cell lines by 70 to 80 percent. All drugs were used at concentrations having little effect on RNA and protein synthesis. However, HU and Ara-C significantly reduced DNA synthesis and cell division in the treated cultures.With 2 Figures