HUMAN HERPESVIRUS 4 (HHV-4) /  EPSTEIN-BARR VIRUS (EBV)
(most material taken with permission from Prof.Dr. Jaap M. Middeldorp et al, Crit Rev Oncol Hematol. (2003) ;45(1):1-36)

Table of contents :


  • Epidemiology
  • Genomics
  • Proteomics
  • Transmission
  • Pathogenesis
  • Symptoms & signs
  • Laboratory examinations
  • Differential diagnosis
  • Therapy
  • Experimental animal models
  • Prognosis
  • Web resources

  • Epidemiology : prevalence > 90%, first cultured in 1964ref
    Genomics : like other herpesviruses, EBV has a toroid-shaped protein core wrapped with DNA, a nucleocapsid, a protein tegument and an outer envelope. The envelope carries Gp spikes of which the most abundant structural glycoprotein (Gp) is Gp350/220. The EBV genome is a linear, double stranded DNA molecule of 172 kb and has reiterated 0.5 kb terminal repeats and a reiterated 3 kb internal direct repeat. The unique sequences also contain several repeat elements, often encoding repeat domains in proteins. The BamHI restriction fragments of the B95.8 laboratory strain genome have been completely sequencedref; EBV genes are named after the BamHI restriction fragment containing the RNA start site and their leftward or rightward transcriptional orientation. BamHI-A being the largest fragment, BamHI-B the second largest, with BALF2 being the second leftward reading frame on the BamHI-A fragment and so on. Subsequent genomic sequencing of additional EBV isolates have revealed the existence of 2 predominant EBV-strains, named A-type and B-type (or type-1 and -2, respectively), with significant sequence polymorphism in the EBNA2 (BYRF1) and EBNA3A-C (BERF1-3) genesref1, ref2 and minor ‘sub-strain’ variations in the EBNA1 (BKRF1), LMP1 (BNLF1), Zebra (BZLF1) and various other genesref1, ref2, ref3. In previous studies the A-type virus was found to prevail in most EBV-associated diseases and showed a more efficient transformation of B-cells in vitroref. The relative oncogenicity and biological behaviour of different EBV-strains and variants remains a matter of debateref1, ref2. Most viral genomes remain in cells as episomes : rarely integrated in cell chromosomes. 3 geographic subtypes of EBV have been identified to date differing by their nuclear antigen EBNA2 :

    EBV genome contains 5 miRNAs : computer predictions of the mRNA targets of the 5 micro RNAs include the tumor suppressor p53, retinoblastoma binding protein 3, transcription factor E2F1, and genes involved in the immune response, such as a BCL8 homolog and the ICOS ligand precursor, a gene expressed in B cells that activates the immune system. The findings could also explain why EBV is linked to cancers such as Burkitt's lymphoma (BL) and Hodgkin's lymphoma (HL). Micro RNAs are expressed quite well in latent infected cells : most EBV genes are not expressed in latent cells—one of the reasons that the virus becomes latent—with the exception of a few latency-associated transcripts (LAT). One of the micro RNAs, miR-BART2, has the potential to target one of the EBV genes that encodes a DNA polymerase : degradation of that transcript could conceivably contribute to either the establishment or maintenance of latencyref. People have found conserved miRNA sequences in animals and humans and plants—and now they're also in viruses.
    Proteomics : the EBV genome contains over 100 open reading frames (ORFs). These are named after the BamHI restriction fragment on which they are located as indicated above. Only about 50–60% of the gene products have been characterized to date and much remains to be learned about their pathogenic role in vivo. A number of technical developments, such as (multi-primed) RT-PCR and NASBA, have allowed a sensitive and reliable documentation of EBV transcriptional activity in most disease syndromesref1, ref2. Transcription of various subsets of these genes has been well documented in EBV+ cell lines and can be detected in cellular samples from patients with a multitude of EBV-associated acute, chronic and malignant disease syndromes. In most EBV proliferative syndromes EBV gene expression is rather limited and does not involve lytic genome replication or production of new virions. In EBV-associated malignancies, the virus genome is present in the individual tumor cells in its latent episomal (closed circular) form and the viral genome is replicated during each cell division by host DNA polymerase together with host chromosomes. The detection of latent episomal EBV-DNA can be used to define tumor and virus clonality and is taken as proof for early involvement of EBV in tumor developmentref1, ref2. Consequently, most EBV disease syndromes are associated with so-called EBV latency. During such latent infections, the virus remains transcriptionally active—albeit in a restricted fashion—and the genes expressed then are referred to as ‘latent genes’
    EBV genes and their (putative) functions

    &z.cirf;, Positive; , negative; &z.cirf;, positive in some cases; ?, not determined.

    Transmission : direct or indirect through oropharynx ("kissing disease"), blood or vehicles. Sexually active teens and adults may be exposed to a larger dose of EBV through particularly "deep" kissing, or possibly through genital fluids, which can carry the virus. Acquisition of EBV is enhanced by penetrative sexual intercourse, although transmission could occur through related sexual behaviors, such as "deep kissing." EBV type 1 infection is significantly more likely to result in IM. A large EBV type 1 load acquired during sexual intercourse can rapidly colonize the B cell population and induce the exaggerated T cell response that causes IM. Thus, IM could, perhaps, be prevented with a vaccine that reduces the viral load without necessarily inducing sterile immunityref. Under normal circumstances, EBV-infection is restricted to humans, although some types of monkeys can be infected experimentallyref. EBV enters epithelial cells in the oropharyngeal mucosa through 3 CD21-independent pathways: Pathogenesis : the finding that individuals with the heritable disorder X-linked agammaglobulinemia harbour no EBV in their blood or throat washings and do not have EBV specific memory cytotoxic T lymphocyte (CTL) response, indicates that B lymphocytes, and not oropharyngeal epithelial cells, are required for primary EBV-infectionref [226]. At present, it is thought that primary EBV-infection occurs in the oropharynx via exchange of cell-free virus or productively infected cells in salivaref [241]. The critical steps underlying the initial EBV B-cell transformation are depicted below :

    Schematic presentation of the initial events leading to EBV-induced transformation of human B-cells. The first steps consist of virion CD21 binding, cell penetration and capsid transport, host cell activation and viral gene expression following release of the linear viral genome in to the cell nucleus. Subsequently, a well-orchestrated series of molecular events is initiated, starting with the expression of EBNA2 and EBNA-LP and leading to the expression of LMP1 the major viral oncogene and multiple host genes as well and to the host-driven circularisation of the viral genome. Finally, expression of EBNA1 is induced allowing the episomal viral genome to replicate with dividing cells, thus completing the growth transformation process. Additional expression of EBERs, BARTs and LMP2a will modify the transformed state but are not essential for this process. The resulting EBV transformed cell will continue to grow indefinitely, expressing the latency-III program. In vivo this program is not tolerated by the immune system and EBV-infected cells will (be forced to) switch-off expression of the major immunogenic proteins. In vivo EBV persists in transcriptionally silenced memory B-cells which only express non-coding EBER1, 2 and BARTs and occasional low levels of LMP2 and EBNA1.

    Schematic representation of EBV RNA expression profiles in different EBV-infected B-cell populations in tonsils and peripheral blood of healthy EBV-carriers and their proposed relationship. Naive B-cells are infected by EB-virions that enter the oropharyngeal lymph nodes by crossing epithelial barriers. EBV glycoprotein gp350 binds to the receptor CD21, present on all B-cells. Under influence of CD21-triggering and the subsequent EBNA2-driven transcription program, naive B-cells differentiate into B blasts that express the full set of latent EBV RNAs and which are probably controlled by anti-EBV cytotoxic T-cell responses. The B blast further differentiates through the germinal center via centroblast to centrocyte. Both cell types express the latent membrane proteins LMP1 and LMP2, which provides them with growth and survival signals in absence of antigen, and EBNA1 which is essential for maintenance of the viral genome in the host cell. Finally, differentiated EBV-infected cells enter the circulation as memory B-cells, that are generally silent for viral RNA expression but may occasionally express LMP2 RNA. Infection of B lymphocytes is mediated through interaction of the viral Gp350/220 with CD21 / CR2 / EBVR, the physiological receptor for the complement factor C3dref1, ref2 [227 and 228]. After CD21 binding the viral envelope fuses with the host cell membrane and triggers host cell activationref1, ref2 [227 and 229]. The nucleocapsid is then transported to the nuclear boundary and subsequently degraded, releasing the linear viral DNA into the nucleus, which then leads to initial EBNA2 and EBNA-LP transcriptionref [39]. EBNA2 and EBNA-LP are essentially required for initial B-cell growth transformation, but their activity is modulated by the subsequent expression of EBNA3A–C. A second essential event is the recircularization of the viral genome at the terminal repeats, for which the host cell DNA repair machinery is usedref1, ref2 [230 and 231]. Although episomal forms are by far prevailing in EBV-associated malignancies in vivo, a small number of studies have described cell lines with integrated EBVref1, ref2, ref3 [232, 233 and 234]. In the first stages after recircularization of the genome, the full spectrum of latent proteins is expressedref [39]. This complex event which leads to efficient growth transformation of the host cell includes the well coordinated transcription of poly-cistronic mRNAs derived from promoters in BamHI-C and -W, encoding EBNA1 together with one or more of the other EBNAs (EBNA2, -3a, -3b, -3c and -LP)ref [39] and transcription of the major EBV oncogene LMP1, encoded in BNLF1. This transcription pattern allows rapid polyclonal expansion of the infected B-cells as lymhoblasts and is, therefore, referred to as the ‘growth program’ref1, ref2, ref3 [171, 235 and 236]. In vivo, incoming EBV predominantly infects subepithelial B-cells to become transformed B-blastsref1, ref2 [39 and 237], but these are rapidly eliminated by the host T-cell response, which is mainly directed against viral lytic genes during early primary infection and against EBNA3a, -3b and -3c during lifelong persistenceref1, ref2, ref3, ref4 [237, 238, 239, 240 and 241]. The fulminant T-cell response against these freshly EBV transformed cells in adolescents and adults is the basis of the mononucleosis syndromeref [241]. Throughout life, outgrowth of EBV transformed B-cells is kept under control by a persistent alert immune response to latency-associated products, especially involving EBNA3A–C and LMP2ref [240]. Part of the EBV-infected lymphoblasts proliferate and differentiate through a germinal center-type reaction and subsequently enter the peripheral B-cell pool as resting memory cellsref1, ref2, ref3, ref4, ref5 [171, 235, 236, 242 and 243]. It is likely that the continuous but limited expression of latent genes associated with the growth program is responsible for maintaining the high level of T-cell memory to these potentially dangerous cells. The pathogenic consequences of failing T-cell surveillance is seen in immunosuppressed individuals who are at high risk of developing lymphoproliferative disease and malignant lymphoma, that are predominantly EBV drivenref [241]. Like other herpesviruses, EBV persists lifelong in its host. Total body irradiation of EBV-seropositive individuals awaiting bone marrow transplantation leads to eradication of the virusref [244]. This indicates that the cellular compartment where the infected cells reside must be associated with the hemopoietic tissue. More recent studies have shown that resting memory B-cells are the reservoir for latently present EBVref1, ref2, ref3, ref4, ref5, ref6 [170, 171, 245, 246, 247 and 248]. In fact, it was already known for many years that it is possible to grow EBV transformed B-cells directly from the blood of virtually all EBV-seropositive individuals, provided that T-cells are depleted or suppressed in vitroref [241]. In vivo, these infected cells can escape CTL-mediated killing because the expression of immunogenic EBV-proteins such as EBNA3a, -3b and -3c is silenced once latent infection has been establishedref1, ref2, ref3, ref4, ref5 [39, 246, 247, 248 and 249]. This silencing is accomplished by methylation of early latency promoters Cp and Wpref1, ref2, ref3 [66, 250 and 251]. Because EBNA1 is indispensible for maintenance of the viral genome in the dividing host cellsref1, ref2 [39 and 77], its transcription is continued, but now initiated from an autoregulated promoter in BamHI-Q (Qp)ref [82]. Interestingly, Qp-driven EBNA1 transcription is found in all EBV-associated malignancies of non-immunocompromized patients. Although EBNA1 is a foreign protein to the host, EBV-infected cells expressing EBNA1 are not killed by CTLs due to the inhibitory effect of the protein's Gly–Ala repeat on proteasomal processing and subsequent MHC class I-restricted presentationref [90]. This is considered to be an important mechanism by which EBV+ tumor cells escape CTL-mediated killingref [249]. Given the fact that memory CTLs reactive against most EBNAs remain clearly detectable for liferef1, ref2, ref3, ref4 [238, 239, 240 and 241], it is generally thought that there must always be a subset of B-cells present that express the growth program. Recently, this subset was shown to consist of naive (IgD+) B-cells in the mantle zones of the tonsilref1, ref2 [246 and 247]. Circulating EBV+ B-cells may express defined homing receptors and cytokine response receptors, that preferentially direct them to the epithelial surfaces in the body, where these cells may be periodically triggered into the lytic cycle in order to maintain shedding of infectious virus in the oropharynxref [241]. It is thought that this switching process is influenced by signals that normally control B-cell behaviour, such as antigen-driven activationref1, ref2, ref3, ref4 [171, 241, 242 and 243]. Indeed careful analysis of the localization of EBV+ B-cells in tonsils and salivary gland epithelia during infectious mononucleosis (IM) and latent carriership have revealed that these cells preferentially locate in the interfollicular region rather than in the germinal center and also accumulate around the crypts and subepithelial layers of the tonsilref1, ref2, ref3 [237, 252 and 253]. The differential expression of defined cytokine receptors, such as CCR6, CCR7, CCR10 and CXCR4 and CXCR5 may be responsible for this phenomenonref [254]. Local antigen triggering and CD40 activation may subsequently lead to virus production and transepithelial secretionref [241]. The persistent (low level) production of infectious virus progeny is reflected by the life-long presence of IgG antibodies to the viral capsid antigen/membrane antigen (VCA/MA) complexref1, ref2 [92 and 255].
    Table 2. EBV latent gene expression patterns in EBV associated disorders

    Table 3. Overview of EBV transcriptiona in EBV associated diseases
    Important factors during virus-induced lymphomagenesis and carcinogenesis are growth transformation in combination with genetic instability, inhibition of apoptosis, angiogenesis and inhibition of (or evasion from) the local immune response. Individual EBV gene products were shown to exert such effects in vitro, and in concerted action are considered to play an important role in the genesis of EBV+ malignancies in vivo. Different EBV gene expression patterns are recognized, which are generally referred to as the latency programs (types) I, II and III. It is conceivable that these expression patterns are subject to the nature of the cells from which the respective malignancies are derived, or that they are in fact subject to the host immune response as is the case in PTLD and ARL. In addition to these well-established gene expression patterns, differential expression patterns were found among other EBV genes that encode homologs to human proteins involved in proliferation, differentiation, apoptosis inhibition and suppression of the local immune response. Some viral latency genes may predominantly provide their function in the initial B-cell transformation, like EBNA2 and EBNA-LP, and are subsequently downregulated or even switched off. Other latency genes, like EBNA1 and LMP2a, may be more essential in maintaining the long-term survival of the EBV genome in a resting cell environment, whereas LMP1 may provide the temporary growth kick when such cells pass through lymph nodesref [171]. The pleiotropic effects of LMP1 expression, together with external growth or differentiation inducing stimuli may well provide the basis for pre-malignant growth. It is fascinating to realize that long-term evolutionary co-existence have thought the virus to evade immune elimination by preventing the recognition of its most essential gene products, especially EBNA1 (via Gly–Ala proteasome inhibition) and LMP1, which is non-immunogenic and has distinct direct and indirect immunosuppressive functionsref1, ref2, ref3 [131, 351 and 437]. As indicated before the well regulated and well balanced, low level expression of LMP1 may be most crucial for persistence of EBV transformed B-cells. When activated in subepithelial layers additional viral genes can be expressed as well providing growth or survival functions. Transcripts encoding BHRF1 (a functional homolog of Bcl-2) were mainly found in B-cell lymphomas (both of immunocompetent and immunocompromized patients)ref1, ref2 [210 and 211]. Weak BHRF1 transcription signals were found in some T-cell lymphomas and HDs, but these signals are ascribed to the presence of EBV-positive reactive cellsref1, ref2, ref3 [54, 210 and 212]. BHRF1 transcription and protein expression at the single cell level in most of the lymphoid disorders has yet to be determined. In NPC the data suggest that BHRF1 expression in NPC is only limited. Preliminary data indicate that BHRF1 protein can also be detected by immunohistochemistry in this disorderref [212]. Particularly strong expression of early BHRF1 transcripts is confined to OHLref1, ref2 [54 and 431]. It is thought that the putative BHRF1 protein may act in addition to the LMP1 induced Bcl-2 protein acts in prevention of host cell apoptosis, enhancing survival of the host cells during production of viral progeny ref1, ref2[438 and 439]. Newly synthesized virions released into the environment or by cell-cell contact with surrounded cells EBV may be transmitted. When the virus then is capable of entering an epithelial cell additional genes, that are not part of the B-cell program may become expressed, one of which is the BARF1 gene. BARF1 was originally recognized as an early transcriptref [189]. Using a reversed northern blotting technique, the transcript was found to be preferentially expressed in epithelial but not in lymphoid cellsref [190]. More recently, using NASBA, we found BARF1 transcription exclusively in NPCs and in OHL but not in lymphoid disordersref1, ref2 ( Fig. 5) [50 and 54]. In addition to these studies, BARF1 transcripts were also detected in EBV-associated gastric carcinomasref [191]. Recently, expression of BARF1 at the protein level was detected in NPC biopsies by immunoblottingref [192]. These findings indicate that BARF1 expression is specific for EBV-associated epithelial malignancies. BARF1 has clear effects on epithelial cells in vitroref [194], in more than one aspect mimicking the role of LMP1 in B-cellsref [440]. Transcription of BCRF1 (a functional homolog of human IL-10) occurs almost exclusively in OHL [54], which represents a productive infection. In this and other productive infections, the BCRF1 protein may play a role in the inhibition of the local immune response. These observations together suggest that multiple products encoded within the EBV genome may exert functions that may be relevant for pathogenic events in vivo. A number of genes with putative interesting functions, such as the cytokine receptor encoded in BILF1, BDLF2, a protein with homology to cyclin-Bref [54] and the BFRF1 putative virion protein, remain to be exploredref [441]. Furthermore, the in vivo expression and functional role (if any) of the intriguing but yet illusive proteins encoded in ORFs RPMS1, A72 and (RK-)BARF0 located within the BARTs that are abundantly transcribed in all EBV-associated malignancies remain to be defined. In addition, the relative importance and interactive collaboration by the individual gene products and the relevance of subtle mutations found in various EBV isolates recovered from directly from tumor tissues or patients with distinct clinical syndromes associated with EBV-infection largely remain to be established. Further unraveling of these viral functions and their ‘well orchestrated (inter)action(s)’ may permit the development of antiviral agents capable of interfering with these functions with options for future therapeutic intervention. EBV and some of its latent gene products have been demonstrated to modulate the expression of various cellular (proto-onco-) genes in vitro. The most obvious way to study the effect of the presence of EBV on the expression of these genes in vivo, is the comparison of their expression in EBV-positive and -negative cases of a single clinical entitiy. In this context, HD is a suitable model, because both EBV-associated HDs and EBV-negative HDs are recognized. Moreover, several findings suggest that EBV-associated and EBV-negative HDs have a different pathogenesis. For example, H-RS cells in EBV-associated HDs express high amounts of MHC class I molecules, whereas MHC class I appears to be dowregulated in EBV-negative HDsref [442]. MHC class II and a number of co-stimulatory molecules as well as intracellular TAPs associated with peptide transport after proteasomal degradation seems to be functionally intactref [362]. This is also found for NPC and T/NK cell lymphomasref1, ref2 [336 and 394]. Most EBV-associated HDs express particularly abundant levels of LMP1 and LMP2, proteins which normally can evoke a CTL response. However, H-RS cells apparently are not killed by CTLsref1, ref2 [363 and 364]. Several underlying mechanisms have been proposed for this phenomenon. It is thought that the presence of the CTLs results in a selection of H-RS cells that are resistant to CTL-mediated (and probably also therapy-mediated) apoptosis. HDs in which this has occurred, are likely to express low amounts of p53 and high amounts of bcl-2 in their H-RS cellsref [368]. However, the expression of p53 (as determined by immunohistochemistry) is not related to the presence of EBV, and the expression of bcl-2 even shows an inverse relation to the presence of EBVref [368]. This indicates that induction of apoptosis resistance by modulation of p53 and bcl-2 expression is probably not a mechanism used by EBV in HD. This may also hold for NPC where high levels of p53 and bcl2 coexist, together with highly expressed PCNAref1, ref2 [391 and 392]. Alternatively, p53 mediated apoptosis may be inhibited by upregulation of A20 expression via LMP1. We tested A20 expression in EBV-positive and -negative HD cases using NASBA, but we could not find a relation between A20 transcription and the presence of EBV (Brink et al., unpublished data). This seems to be in agreement with the finding that EBV apparently does not interfere with the normal function of p53 in HDref [204]. It would be best to confirm these data morphologically, but at present no antibodies are available that recognize human A20 in clinical material. To prevent CTL-mediated killing of EBV-infected H-RS cells, inhibition of the local CTL response by EBV is also a possible mechanism. EBV-associated HDs express significantly higher amounts of human IL-10ref1, ref2, ref3 [161, 362 and 443], which may contribute to immune evasion. We have shown that HDs do not express the viral homolog of Interleukin-10 (BCRF1) but that the detected IL-10 expression is of host cell originref [161]. Upregulation of human IL-10 upon EBV-infection in vitro has been demonstratedref [365], and, interestingly, elevated human IL-10 expression levels have been detected in the H-RS cells of EBV-positive HDsref1, ref2, ref3 [161, 362 and 443].
    Table 6. Overview of cellular genes and their functions of which expression and/or function is modulated by EBV

    None: no differences found for EBV+ and EBV? cases.

    Using RT-PCR, we have also analyzed the expression of the proto-oncogene c-fgr. C-fgr was shown by others to be upregulated upon EBV-infection, and EBNA2 is most likely the EBV gene to induce c-fgr expressionref1, ref2 [96 and 444]. Moreover, alternative splicing of c-fgr transcripts was reported in EBV-positive cell linesref [445]. Using RT-PCR, we have performed a qualitative analysis of c-fgr transcription in EBV-positive (n=4) and -negative HDs (n=4), in EBV-associated PTDLs (n=3) and ARLs (n=4), and one IM case. We found no clear relation between the presence of EBV and c-fgr transcription in HD (Brink et al., unpublished data). Moreover, c-fgr RT-PCR signals in HD were relatively weak compared with positive controls. In most PTLDs and ARLs we found transcription of c-fgr, including the EBV specific alternative transcript. These data suggest that upregulation of the c-fgr proto-oncogene is not a mechanism used by EBV in HD, but may play a role in PTLDs, ARLs and IM. Moreover, these data strengthen the finding that EBNA2 (which is expressed to some extent in PTLDs, ARLs and IM but not in HD) is the EBV gene responsible for c-fgr upregulation. Future studies should aim to investigate c-fgr protein expression at the single cell level. In conclusion, differences in EBV gene expression patterns exist between the various EBV-associated diseases. This is true for lymphomas of immunocompromized versus immunocompetent patients, for lymphomas of B-cell versus T-cell origin, and most strikingly for lymphomas on the one hand and epithelial disorders on the other. For some of the differentially expressed genes, such as BARF1, it remains to be investigated whether the observed expression pattern is subject to host cell regulation factors or contributes actively to differences in pathogenesis. Therefore, future studies should not only aim to determine EBV gene expression in EBV-associated diseases, but should also include more fundamental research on expression regulation and functional interactions. Moreover, it is important to notice that high mRNA expression does not always coincide with expression at the protein level; we have shown this for BARF0 but it may well be that the EBV genome gives rise to other non-translated transcripts. In the future, it would be worthwhile to develop additional antibody reagents against the various (putative) EBV-proteins and use these for in situ expression analysis, in combination with mRNA profilingref. EBV-induced gene 3 (EBI3) is expressed in DCs and is part of the cytokine IL-27ref that controls Th cell development. However, its regulated expression in DCs is poorly understood. EBI3 is expressed in splenic CD8-, CD8+, and plasmacytoid DC subsets and is induced upon TLR signaling. Cloning and functional analysis of the EBI3 promoter using in vivo footprinting and mutagenesis showed that stimulation via TLR2, TLR4, and TLR9 transactivated the promoter in primary DCs via NF-kB and Ets binding sites at -90 and -73 bp upstream of the transcriptional start site, respectively. Furthermore, NF-kB p50/p65 and PU.1 were sufficient to transactivate the EBI3 promoter in EBI3-deficient 293 cells. Finally, induced EBI3 gene expression in DCs was reduced or abrogated in TLR-2/TLR4, TLR9, and MyD88 knockout mice, whereas both basal and inducible EBI3 mRNA levels in DCs were strongly suppressed in NF-kB p50-deficient mice. In summary, EBI3 expression in DCs is transcriptionally regulated by TLR signaling via MyD88 and NF-kB. Thus, EBI3 gene transcription in DCs is induced rapidly by TLR signaling during innate immune responses preceding cytokine driven Th cell developmentref. Increased numbers of EBV-infected cells in areas of active inflammatory bowel disease are secondary to influx or local proliferation of inflammatory cells & do not contribute significantly to local production of EBI3ref
    Release of progeny virions from polarized cells occurs from both their apical and basolateral membranes. No CPE, no productive replication in vivo, immortalization of B lymphocytes in vitro. Polyclonal activation of B cells => heterophilic IgMs.
    Expression of IL-7Ra was lost from all CD8+ T cells, including EBV epitope-specific populations, during acute infectious mononucleosis (IM). Thereafter expression recovered quickly on total CD8+ cells but slowly and incompletely on EBV-specific memory cells. Expression of IL-15Ra was also lost in acute IM and remained undetectable thereafter not just on EBV-specific CD8+ populations but on the whole peripheral T and NK cell pool. This deficit, correlating with defective IL-15 responsiveness in vitro, was consistently observed in patients up to 14 years post-IM but not in patients after CMV-associated mononucleosis, nor in healthy EBV carriers with no history of IM, nor in EBV-naive individuals. By permanently scarring the immune system, symptomatic primary EBV infection provides a unique cohort of patients through which to study the effects of impaired IL-15 signalling on human lymphocyte functions in vitro and in vivoref.

    Symptoms & signs :

    Laboratory examinations : Differential diagnosis with other atypical lymphocytoses. A lymphocyte–white blood cell count (L/WCC) ratio > 0.35 had a specificity of 100% and a sensitivity of 90% for the detection of mononucleosis and against acute purulent tonsillitisref.
    Therapy : Patients given ampicillin or amoxicillin develop a rash (a.k.a. Dave Rouse disease)
    Except for OHL, which is benign and represents a purely lytic infection with large amounts of EBV replicating freely in the lesions, all the other EBV diseases are malignancies characterized by latent infection that relies on cellular enzymes for EBV episomal DNA synthesis. Therefore, it is not expected that antiviral drugs directed at viral synthetic processes would affect EBV in the latent phase. In latent infection, the linear double-stranded genomes characteristic of productive lytic infection and encapsidated in virus particles are not made. Rather infection persists via controlled replication of viral episomes that are found only in the nucleus in a nucleosomal form. These circular supercoiled genomes are replicated once during cell division and perpetuated in progeny cells indefinitely. Replication is mediated by host DNA polymerase (and other enzymes of the cellular replicative machinery). Episomal copy number is tightly regulated and remains constant while expression of viral genes is greatly limited to several latency genes. None of these processes is affected by, or sensitive to, antiviral drugs to any degree, as shown by their lack of effect on latently EBV-infected cell lines or tumours (Pagano JS. Epstein-Barr virus: Therapy of active and latent infection. In: Jeffries DJ, De Clercq E, eds. Antiviral Chemotherapy. John Wiley & Sons Ltd. 1995; pp. 155–95). A possible exception might be the initially polyclonal EBV lymphoproliferative disorders, in which occasional cells do exhibit lytic rather than latent infection; in such cases, theoretically antiviral therapy might prevent secondary infection of a new population of B cells. There is only anecdotal evidence for such an effect clinically, and it is scant. However, Chodosh et al. have reported experiments in which treatment of latently EBV-infected cells with hydroxyurea led to loss of episomesref, and in a subsequent case report detailed apparently successful use of hydroxyurea in a patient with an EBV-related CNS lymphomaref. It should be noted though, that a number of groups are intensively studying means to induce a switch from latent to cytolytic infection as a therapeutic approach for the treatment of EBV+ neoplasms, the rationale being that viral replication causes cytolysis of the infected cells and could do so in tumoursref : ... all of which alone or in combination induce viral reactivation. Combinations of such inducers with ganciclovir, which is activated by EBV PK (or TK) and thus toxic for infected and some neighbouring cells, has led to some progress in treatment of EBV+ malignancies in animal modelsref1, ref2, ref3 and in Phase I/II clinical trialsref. In quite another experimental approach to treatment of EBV-associated malignancies, phosphonated nucleoside analogues have been used to cause apoptosis of human EBV+ anaplastic or undifferentiated nasopharyngeal carcinoma (NPC) / lymphoepithelioma grown in athymic mice. Since NPCs are latently infected, again the antitumour effect must be independent of the antiviral effects of these drugsref1, ref2 (Wakisaka N, Yoshizaki T, Murono S et al. Ribonucleotide reductase inhibitors enhance cidofovir-induced apoptosis in EBV+ nasopharyngeal carcinoma xenografts. Int J Cancer 2005; 116: in press). Interestingly, cidofovir has been reported to produce complete responses in vivo in another virus-associated neoplasm, laryngeal papillomatosis, which is caused by human papillomavirusref (Snoeck R, Andrei G & De Clercq E. Cidofovir in the treatment of HPV-associated lesions. Verh K Acad Geneeskd Belg 2001; 63: 93–120, discussion 120–2). Such effects of cidofovir might be linked to decreased expression of EBV LMP1 and consequent aberrations in apoptotic mechanismsref, but there is evidence that weighs against these conclusionsref, and so how these nucleotide analogues affect neoplastic growths is far from clear and remains an important topic for continued studyref
    Experimental animal models : at present it is not known to what extent the mechanisms described in vitro also play a role in vivo. To gain more insight into the in vivo situation, several animal models have been developed. The pathogenic aspects of separate EBV gene products have been studied by means of transgenic mice. These studies clearly indicate putative pathogenic roles for EBNA1ref [87], LMP1 (both in lymphoidref [47] and epithelialref [48] tissue), and LMP2Aref [168] in driving oncogenic cell transformation and inhibition of cellular differentiation. However, the implications of these findings for the pathogenesis of EBV-infection and malignant transformation in humans, where EBV persists in the face of a highly responsive immune system, remain to be clarified by further studies. In addition, animal models have been developed to study the biology of EBV-infection. Especially cottontop tamarins are susceptible for EBV-infection: administration of a high intravenous dose of the EBV B85-8 strain rapidly produces gross, multiple, malignant lymphomas in 100% of these animalsref [418]. However, this pathogenesis is clearly not representative for the situation in humans, where several types of lymphomas develop only after a long period of latent EBV-infection. A more closely related animal model was developed by Wedderburn et al. using the common marmoset which developes a persistent infection more closely resembling EBV-infection of humans. However, only limited experience exists in this modelref [419]. From lymphoma tissue arising in various monkey species EBV-like viruses have been recovered that have subsequently been used to develop an animal model for lymphomagenesisref1, ref2, ref3, ref4 [420, 421, 422 and 423]. An excellent alternative model was developed by Moghaddam et al.ref [424], who did not use EBV but a naturally endemic rhesus lymphocryptovirus. This animal model represented several aspects of human EBV-infection, including oral transmission, atypical lymphocytes, lymphadenopathy, latent infection in peripheral blood and serologic responses to latent and lytic viral antigens. Interestingly, tamarins were shown to be effectively protected from lymphomas after vaccination with a gp340 vaccine containing the native fully glycosylated Gpref [425], although in protected animals virus-carrying cells could still be detected which showed a restricted gene expression patternref [426]. Similar studies have recently been undertaken in the common marmoset, showing reduced shedding by EBV challenge in gp340 immunized animalsref [427]. In humans, during natural infection gp340 also elicits virus-neutralizing antibodies which remain present in healthy virus carriers throughout liferef [428]. Indeed a small human trial with recombinant vaccinia virus expressing gp340 showed promising results in delaying primary infection in virus-naïve infantsref [429]. In addition to studies in monkeys, EBV-mediated lymphomagenesis has also been studied in SCID mice and in nude miceref1, ref2, ref3 [430, 431 and 432]. This model also proved a valuable tool for anti-tumor therapeutic studies of EBV lymphomasref1, ref2 [433 and 434] and carcinomasref1, ref2 [435 and 436]. It is, however, difficult to extrapolate these data to EBV-mediated lymphomagenesis or carcinogenesis in humans, because these nude mice and SCID mice lack (part of) the immune responses which play a role in the establishment and control of EBV-infection in humans.
    Prognosis : establishes a life-long persistent infection in > 90% of the world's population
    Web resources : International Association for Research on EBV and Associated Diseases

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