Showing papers on "Gammaherpesvirinae published in 1995"

Evidence for lytic infection by Epstein-Barr virus in mucosal lymphocytes instead of nasopharyngeal epithelial cells in normal individuals

Abstract: Normal nasopharyngeal tissues from 23 individuals who died of causes unrelated to the upper respiratory system and had no evidence of Epstein-Barr virus (EBV)-related diseases were studied using in situ hybridisation (ISH) and immunohistochemistry for the detection of EBV RNA and expression of EBV proteins, respectively. ISH using 35S-labelled riboprobe for EBV EBER RNA showed occasional to a few EBER + lymphocytes in the stroma of nasopharyngeal mucosa in 14/16 cases with available paraffin-embedded tissues. In addition, very rare intraep-ithelial EBER+ lymphocytes were also detected in 3/16 cases. However, in none of these cases was EBER detected in the epithelial cells. Similar results were obtained using a nonradioactive ISH method for EBER (Dako). In 3/23 cases, im-munostaining using monoclonal antibodies for EBV proteins on cryostat sections showed occasional cells in the stroma expressing EBV nuclear antigen 2 (EBNA2), latent membrane protein-1 (LMP), and switch protein encoded by BZLF1 gene (ZEBRA) in two cases and only very rare LMP+ and ZEBRA+ cells in one other case. Double immuno-staining combining alkaline phosphatase anti-alkaline phosphatase (APAAP) for CD markers and indirect immunofluorescence for LMP showed that the LMP+ cells were either CD19+ or less frequently CD3+, but none were CD68+. These results show that both B and T lymphocytes harbouring EBV can be found in the normal nasopharyngeal tissues. Interestingly, EBV proteins associated with lytic viral replication—diffuse early antigen (EA-D), viral capsid antigen (VCA), or membrane antigen (MA)—were also detected in rare cells in the stroma in one case, and in another case only one MA+ cell was detected. These results provide evidence for a lytic type of infection by EBV in the normal nasopharynx involving a very small proportion of stromal lymphocytes and support the view that nasopharyngeal lymphocytes can act as a reservoir for EBV in normal individuals. © 1995 Wiley-Liss, Inc.

Absence of Epstein-Barr Virus in Medullary Carcinoma of the Breast as Demonstrated by Immunophenotyping, In Situ Hybridization and Polymerase Chain Reaction

Abstract: Medullary carcinoma of the breast is an epithelial malignant proliferation that shares many characteristics (macroscopic, microscopic, epidemiologic, and prognostic) with lymphoepithelioma-like carcinomas of various sites. The authors hypothesized that they could also share the same etiologic agent, the Epstein-Barr virus (EBV). Epstein-Barr virus, a virus of the herpesvirus family, is to be associated with lymphoepithelioma-like carcinomas of the nasopharynx, stomach, lung, thymus, and salivary gland. Therefore, the authors looked for the virus in a series of 10 medullary carcinomas of the breast. Using immunohistochemistry, in situ hybridization and polymerase chain reaction, this investigation failed to show evidence of EBV. Similar negative results have been reported in lymphoepithelioma-like carcinomas arising in the skin and in the uterine cervix, which like the breast do not originate in the foregut. These results suggest that the pathogenesis of these tumors is not unique, implicating probably different etiopathogenic entities. (Key words : Epstein-Barr virus ; Lymphoepithelioma-like carcinoma ; Medullary carcinoma of the breast) Am J Clin Pathol 1995 ;103 :449-452.

The expression of Epstein-Barr virus latent proteins is related to the pathological features of post-transplant lymphoproliferative disorders.

Abstract: Transplant recipients are at increased risk for the development of post-transplant lymphoproliferative disorders (PTLDs). PTLDs harbor genomes of the Epstein-Barr virus, a herpesvirus that immortalizes B cells in vitro. At least five viral proteins are required for immortalization. Two of them are particularly important. Latent membrane protein (LMP) has transforming activity in fibroblasts, and Epstein-Barr antigen (EBNA)2 transactivates the expression of numerous cellular and viral genes. To determine whether the expression of EBNA2 and LMP is related to the histological and clinical presentation of PTLD, we tested their expression in 14 Epstein-Barr virus-positive cases. Using monoclonal antibodies to EBNA2 and LMP on paraffin sections, we found an expression of both proteins in 2 of 3 polymorphic PTLD and in 7 of 8 cases of monomorphic, large cell PTLD, without plasmacytic differentiation. One polymorphic and one large cell PTLD expressed LMP only. LMP and EBNA2 were found particularly in immunoblasts. The number of positive cells was extremely variable in the different cases as well as within the same biopsy. Three cases of PTLD had morphological and phenotypical features of plasmacytomas and did not stain for EBNA2 or LMP. This suggests that the expression of EBNA2 and LMP is related to the differentiation stage of the infected cells and that other viral or cellular proteins may contribute to tumor growth.

Virus-associated hemophagocytic syndrome characterized by clonal Epstein-Barr virus genome

Abstract: Virus-associated hemophagocytic syndromes are a heterogeneous group of disorders in which viral infection is associated with a proliferation of hemophagocytic histiocytes throughout the reticuloendothelial system. The authors report the case of a 24-year-old Vietnamese male who developed a hemophagocytic syndrome associated with Epstein-Barr virus (EBV) and who died following a rapidly progressive course. A proliferation of reactive-appearing lymphoid cells was associated with an extensive proliferation of erythrophagocytic histiocytes. Immunophenotypically, the lymphoid infiltrate consisted of CD56+ natural killer cells, predominantly CD8+ T-cells and rare B-cells (CD20+). Double-label immunohistochemical studies showed CD3+ T-cells and CD56+ natural killer cells to be distinct cell populations. Combined immunohistochemical-in situ hybridization studies localized EBV to CD43+, CD3-, CD68-, lymphoid-appearing cells, indicating the presence of EBV within natural killer cells. Southern hybridization analysis of EBV genomic termini revealed clonal EBV genome. However, there was no detectable immunoglobulin or T-cell receptor gene rearrangements. The findings indicate that this case of hemophagocytic syndrome represents a clonal proliferation of natural killer cells containing EBV and highlights the importance of the analysis of EBV genomic termini for determination of clonality in EBV-associated proliferations. It is possible that other cases of fulminant EBV-associated hemophagocytic syndromes represent clonal natural killer cell proliferations.

Human herpesvirus 6 variant A, but not variant B, infects EBV-positive B lymphoid cells, activating the latent EBV genome through a BZLF-1-dependent mechanism.

Abstract: Human herpesvirus 6, a predominantly T lymphotropic virus, has been recently shown to infect some EBV-positive B cell lines, and to induce in them the activation of the EBV lytic cycle. Here we have confirmed and extended such observations, showing that (1) this phenomenon is restricted to the variant A of HHV-6: in fact two isolates belonging to the HHV-6 variant B (BA92 and Z29) were neither able to infect any B cell line, independently of the EBV status, nor to induce the EBV genome expression. The only exception is represented by the P3HR1 cells, in which, however, the infection by the variant B does not determine induction of EBV antigens; (2) the presence of the EBV genome contributes to the susceptibility of the B cell lines to HHV-6 infection, increasing the binding sites and the percentage of infectable cells, as detected by immunoelectron microscopy; and (3) HHV-6 infected T cells, transfected with plasmids bearing the promoter regions of the EBV early genes BZLF1 and BMRF1, show a strong transactivation of these promoters.

Squamous Epithelial Proliferative Lesions Associated with Rhesus Epstein-Barr Virus in Simian Immunodeficiency Virus-Infected Rhesus Monkeys

Abstract: Proliferative lesions were found on the squamous epithelium of the tongue, esophagus, or penis or haired skin of the lip, hand, or thorax of 8 simian immunodeficiency virus-infected rhesus monkeys that died of simian AIDS. The lesions were focal and consisted of hyperkeratosis, parakeratosis, and acanthosis in the skin, with additional ballooning degeneration in the tongue, esophagus, and penis. The epithelial surfaces were frequently colonized by Candida species or gram-positive cocci. Intranuclear inclusion bodies were seen in cells in the middle and superficial layers. Herpesvirus virions were found in inclusion-bearing cells by transmission electron microscopy. An Epstein-Barr-like virus was identified in inclusion-bearing cells by immunohistochemistry and in situ hybridization. No virus was detectable in basal layers of the epithelium. These lesions resemble oral hairy leukoplakia in AIDS patients and may thus provide a useful primate model to study permissive epithelial infection by Epstein-Barr-like viruses.

Chronic active Epstein-Barr virus disease in a case of persistent polyclonal B-cell lymphocytosis.

Abstract: Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare haematological disorder. It is characterized by activated and morphologically atypical B lymphocytes and polyclonal IgM production and has been associated with female sex, cigarette smoking, and HLA-DR7 expression. We report a case of PPBL with intermitting symptoms compatible with a chronic fatigue syndrome, recurrent erythema nodosum and multiforme. Serological findings suggested a chronic active Epstein-Barr virus (EBV) infection. Messenger RNA of EBV immediate early gene transactivation BZLF1 was detected in peripheral blood lymphocytes by reverse transcriptase PCR indicating a persistent replication of the virus. Over 2 years of observation we detected varying numbers of atypical lymphocytes. These cells hybridized with a probe specific for the EBV internal repeat region (BamHI W) which indicates a productive infection. Of interest, no reaction was observed with a probe specific for the latency-associated small RNAs (EBERs). The immunological phenotype of the polyclonal B cells was similar to B-cell lines immortalized by EBV in vitro, expressing a number of activation molecules (CD23, CD25, CD54) and the bcl-2 protein. In summary, our findings suggest that persistent EBV replication might be crucial in the development of lymphoproliferative disorders such as PPBL.

Expression of bcl-2 protein and transcription of the Epstein-Barr virus bcl-2 homologue BHRF-1 in Hodgkin's disease: implications for different pathogenic mechanisms.

Abstract: Epstein-Barr virus (EBV) is frequently found in Hodgkin and Reed-Sternberg cells in Hodgkin's disease. Epstein-Barr virus has transforming properties in vitro and might be involved in the pathogenesis of certain types of Hodgkin's disease. One of the possible mechanisms is the upregulation of the human proto-oncogene bcl-2 by the latent membrane protein 1 of EBV in vitro. Another possibility might be the expression of the viral 'bcl-2 homologue', BHEF-1. In the present study of 64 cases of Hodgkin's disease we investigated the expression of bcl-2 at the protein level in relation to the presence of EBV. Moreover, in 10 EBV positive cases we investigated, the expression of the bcl-2 homologue, BHRF-1, by reverse-transcriptase PCR. bcl-2 was detected in 14 of 22 (64%) EBV positive and in 3 7 of 42 (88%) EBV negative cases. In 17 of 22 (77%) EBV positive cases Reed-Sternberg cells were negative (n = 8) or expressed the bcl-2 protein in a very low percentage ( 50%) of the neoplastic cells were bcl-2 positive. Using the reverse-transcriptase PCR with primers amplifying transcripts of BHRF-1 we were able to detect BHRF-1 transcripts in only one of the 10 tested cases of EBV positive Hodgkin's disease. Our data indicate that in EBV positive Hodgkin's disease growth advantage of Reed-Sternberg cells is not obtained by upregulation of bcl-2 or by the EBV homologue BHRF-1.

Induction of CD44 expression by the Epstein-Barr virus latent membrane protein LMP1 is associated with lymphoma dissemination

Abstract: Epstein-Barr Virus (EBV) is implicated in the pathogenesis of endemic Burkitt's lymphoma (BL), B-cell lymphomas occuring under immunosuppression, nasopharyngeal carcinoma and Hodgkin's disease. Two distinct patterns of latent EBV gene expression occur in EBV-associated lymphomas. BLs typically display expression of the nuclear antigen EBNAI only, whereas EBV-associated, non-Burkitt B-cell lymphomas express at least 9 latent viral genes (6 EBNAs and 3 latent membrane proteins), reminiscent of in vitro EBV-immortalized lymphoblastoid cell lines (LCL). BLs are characterized by local, extra-nodal growth, whereas EBV-associated B-cell lymphomas often disseminate to peripheral lymphoid tissue. We show here that BL cells forming local tumors after xenotransplantation into SCID mice disseminate to lymphoid tissue following introduction of the latent membrane protein 1 (LMP1) gene. Introduction of LMP1 into BL cells induced expression of CD44 on the cell surface, a molecule implicated in enhanced lymphoid tumor growth and dissemination. Introduction of CD44 into LMP1-/CD44-BL cells was observed to confer the disseminated tumor growth pattern associated with LMP1 expression. Taken together our results show that expression of LMP1 may regulate expression of CD44 and play an important role in the behavior of EBV-based lymphomas. © 1995 Wiley-Liss, Inc.

Characteristics of EBV-infected cells in HIV-related lymphadenopathy: implications for the pathogenesis of EBV-associated and EBV-unrelated lymphomas of HIV-seropositive individuals.

Abstract: The present study was performed with the aim of better defining the possible role of Epstein-Barr-virus (EBV)-infected cells in the pathogenesis of HIV-related lymphadenopathy syndrome (LAS). In addition, since LAS has been considered as a pre-lymphomatous lesion, we also wished to elucidate the possible contribution of EBV-carrying cells present in LAS tissues to the development of HIV-associated malignant lymphomas. To this end, we have characterized EBV-infected cells in LAS lymph nodes in terms of EBV DNA prevalence, tissue distribution in relation to HIV-carrying cells, virus sub-type, expression of latent and replicative antigens, and presence of clonal EBV episomes. When compared with HIV-unrelated lymphadenopathies (4/10, 40%), LAS showed a higher prevalence of EBV DNA (14/20, 70%). Comparable values of EBV prevalence were detected in LAS with follicular hyperplasia (12/16, 75%) and with follicular involution (4/4, 100%). All EBV+ non-neoplastic lymph nodes from HIV-seronegative patients carried type-I EBV, whereas LAS specimens showed almost equivalent distribution of the 2 EBV sub-types. Of the 14 EBV-carrying LAS, 4 (29%) were positive by Southern-blot analysis for the BamHI-W region of the virus genome but negative for the presence of monoclonal EBV episomes. In situ hybridization revealed a remarkably higher load of EBV-infected cells in LAS than in HIV-unrelated lymphadenopathies. In LAS lymph nodes, EBV-carrying cells were identified as isolated, cytologically normal elements, sometimes with immunoblastic morphology, usually scattered throughout the interfollicular areas. By contrast, the expression of HIV p24 was restricted to germinal center cells. All the EBV+ LAS samples were negative for the expression of EBV-encoded latent (LMP-1 and EBNA-2) and replicative proteins (BZLF-1, BHLF-1, EA-D, EA-R and VCA). In addition, amplification of the immunoglobulin heavy-chain genes using 2 different polymerase-chain-reaction protocols showed evidence of B-cell clonal expansion in 2/20 (10%) LAS, one EBV- case, and one sample with low numbers of EBV-infected cells. These results suggest that (i) EBV-carrying cells are probably not involved in the development of LAS, either directly or indirectly; (ii) type-2-EBV-infected cells are present in LAS lymph nodes from the early phases of HIV infection; (iii) EBV-carrying LAS per se probably does not constitute a lesion at high risk for subsequent development of EBV+ lymphomas; (iv) it is unlikely that a high viral load or strong EBV-mediated antigenic stimulation plays a contributory role in the development of EBV-unrelated lymphomas of HIV-seropositive individuals.

Absence of in situ hybridization evidence for latent- or lytic-phase Epstein-Barr virus infection of preinvasive squamous lesions of the cervix.

Abstract: To investigate whether Epstein-Barr virus (EBV) infection of the uterine cervix plays a significant role in cervical carcinogenesis, 30 preinvasive squamous lesions were subjected to in situ hybridization for (EBER-1,-2, and BHLF1) EBV transcripts which are expressed in latent and lytic infection, respectively. Twenty cases were known to contain EBV sequences by previous polymerase chain reaction (PCR) analysis. Irrespective of EBV PCR status or histological grade, none of the 30 cases demonstrated EBV transcripts in squamous epithelial cells. Two cases showed very occasional EBER-positive stromal cells, most probably representing resident cervical lymphocytes. These findings suggest that EBV plays no part in early cervical carcinogenesis.

Malignant lymphomas induced by an Epstein-Barr virus-related herpesvirus from Macaca arctoides--a rabbit model.

Abstract: Animal models for Epstein-Barr virus (EBV) are restricted to some species of new-world monkeys which develop malignant lymphoid tumours or benign lymphoproliferative diseases after virus inoculation. Similar pathological features were induced in rabbits by the EBV-related herpesvirus ofMacaca arctoides (HVMA). In this study 17 of 32 rabbits infected with varying amounts of HVMA produced from MAL-1 cells developed lymphoproliferative disorders. In 13 rabbits high-grade malignant lymphomas were detected, 4 rabbits revealed the histopathological feature of lymphoid hyperplasia. These lympho-proliferations were shown to be associated with HVMA by PCR and by the expression of EBV-like RNAs (EBER) in 14 and 10 cases, respectively. The homology in the polymerase gene region between DNA from EBV and HVMA, and from HVMA and the malignant tissue was found to be 94.8% and 100%, respectively. All the infected animals produced antibodies to antigens corresponding to early and late EBV proteins. By studying the HVMA expression in MAL-1 cells EBV-like proteins expressed in latency (EBNA1 and EBNA2) and in the lytic cycle (VCA, EA) were detected. Our findings suggested that HVMA caused a symptomatic infection in rabbits with pathological features that fit the conditions of an animal model suitable for testing antiviral drugs and vaccines against EBV.

Herpes viruses in multicase families with rheumatoid arthritis

Abstract: Objective. To investigate the rate and extent of infection by Epstein-Barr virus (EBV), cytomegalovirus, and herpesvirus 6 in families (affected and nonaffected members) with multiple cases of rheumatoid arthritis (RA). Methods. Viral DNA was detected by polymerase chain amplification in cells from saliva and peripheral blood. Human leukocyte antigen pedigrees were characterized. Results. Viral DNA, particularly EBV, was detected in increased frequency (p = 0.029) in the patients with RA compared to their nonaffected relatives. Conclusion. We suggest that in RA multicase families, increased frequency of viral infection is likely a consequence of the disease state and/or due to gene(s) as yet unidentified.

Epstein-Barr virus and brain lymphomas.

Abstract: In 1958, Dennis Burkitt a British surgeon practicing in Uganda reported a high incidence of a tumor of the jaw in African children [1]. Microscopically, the tumor referred to as 'Burkitt's lymphoma' (BL), consisted of small lymphocytic cells with macrophages ingesting lymphocytes giving the classical 'starry sky' appearance [2]. Because the geographical distribution of this tumor was affected by climate factors, an environmental or infectious etiology was sought with little success initially [3, 4]. In 1964, Epstein and Barr were able to grow continuous lymphoblast cell lines in tissue culture from BL tumors [5]. They also were able to demonstrate viral particles in these cell lines by electron microscopy thus supporting the possibility of an infectious etiology [5]. Others were able to detect viral antigens in the nuclei of BL tumor cells as well as their nuclear and cytoplasmic cell membranes [6-9]. A virus was subsequently isolated from this tumor and was referred to as the Epstein-Barr virus (EBV) which was recognized as the agent responsible for infectious mononucleosis [10, 11]. The virus which consists of 172 Kbp double stranded DNA structure was isolated, cloned and completely sequenced in 1984 [12, 13]. The EBV genome consists of five unique regions and a varying number of repeat units [14]. It is classified as Human Herpes Virus number 4 with man as its only reservoir [15]. Biological consequences of E B V infection

Induction of a herpesvirus saimiri small RNA AU binding factor (AUBF70) activity and lymphokine mRNAs by T cell mitogens

Abstract: Herpesvirus saimiri (H. saimiri) can transform T lymphocytes and cause lymphoid tumors in rabbits and New World monkeys. H. saimiri-immortalized T cells express IL-2 and IL-4. The putative oncogenes of a group C strain of H. saimiri have been mapped to a region of the unique L-DNA which includes genes encoding four U-like small nuclear RNAs (HSUR1-HSUR4). Jurkat T cells express a 70 kD RNA binding factor (AUBF70) which binds HSUR2. Here we examined AUBF70 expression in resting and mitogenstimulated human peripheral blood T cells and its sequence specificity and subcellular distribution. Band-shift assays demonstrated that resting human T cells express low amounts of AUBF70 which is induced by mitogen treatment. IL-2 and IL-4 mRNAs were co-induced with AUBF70 suggesting that AUBF70 is a positive regulator of lymphokine gene expression. Normal resting, mitogen-stimulated, and leukemic Jurkat T cells all express AUBF70 with virtually identical V8 proteolytic enzyme digestion patterns. Northern blots demonstrated that HSUR1 and HSUR2 are localized both in the nucleus and cytoplasm. HSUR2 accumulate in the cytoplasm in the presence of actinomycin D, which is consistent with re-transport of HSURs to the nucleus by (an) unstable factor(s). We hypothesize that HSUR1 and 2 transport AUBF70 from the cytoplasm to the nucleus; in the nucleus, AUBF70 binds and stabilizes lymphokine transcripts. Increased stability of lymphokine mRNAs could contribute to oncogenic transformation induced by H. saimiri.

Identification and analysis of an alcelaphine herpesvirus 1 (AHV-1) cDNA clone expressing a fusion protein recognized by AHV-1-neutralizing antisera.

Abstract: Rabbit antiserum to psoralen-inactivated alcelaphine herpesvirus 1 (AHV-1) virions was shown to react specifically with AHV-1-infected cells by indirect immunofluorescence. Western blot analysis using this antiserum identified a 15-kD virion protein that was also detected in infected-cell proteins between 12 and 144 h p.i., and a 37-kD protein present in infected cells between 24 and 120 h p.i. A cDNA library was constructed using mRNA obtained from AHV-1-infected fetal mouflon sheep kidney (FMSK) cells at 48 h p.i., when infected-cell proteins detected by antiserum were in abundance. Screening of the library with the rabbit anti-AHV-1 serum identified several positive clones. Southern blot analysis showed that one clone, designated 8'a, hybridized to a 4.4kbHindIII fragment of AHV-1 DNA. This AHV-1 cDNA clone expressed a fusion protein that was recognized by serum from a naturally and asymptomatically infected white-bearded wildebeest (Connochaetes taurinus albojubatus). The insert was sequenced and found to contain 833 bp. A search of the GenBank database for related sequences revealed greater than 40% homology to several other gammaherpesviruses: herpesvirus saimiri, cottontail herpesvirus, and Epstein-Barr virus.