(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 :

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

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Epidemiology : prevalence > 90%, first cultured in 1964^ref
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 sequenced^ref; 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) genes^{ref1, ref2} and minor ‘sub-strain’ variations in the EBNA1 (BKRF1), LMP1 (BNLF1), Zebra (BZLF1) and various other genes^{ref1, 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 vitro^ref. The relative oncogenicity and biological behaviour of different EBV-strains and variants remains a matter of debate^{ref1, 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 :

• EBNA2 AC strains predominate in Asia
• EBNA2 AD strains predominate in the USA
• EBNA2 B strains have all been identified in black Africa.

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 latency^ref. 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 syndromes^{ref1, 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 development^{ref1, 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.

• latent gene products : comprise about 10 different gene products that are expressed in different transcription profiles (latency types). During latency, EBV expresses a limited number of viral genes, which are involved in tasks such as stimulating cell proliferation, inhibiting apoptosis, blocking viral lytic replication, and assuring accurate and equal partitioning of the episomal viral genome to daughter cells. Because most EBV-linked syndromes are associated with viral latency, the functions of latent gene products have been studied most extensively
• EBV-encoded RNAs (EBER1 and EBER2) are small, non-polyadenylated RNAs with an estimated abundance of 10⁵–10⁷ copies per cell that have a stable secondary structure with extensive intramolecular base-pairing and some homology to tRNAs^{ref1, ref2, Arrand , JR, 145–149}. They are localized in the nucleus, where they are associated with the cellular La antigen^{Clemens, ML, 107–111} and the EBER-associated protein^ref. It has been proposed that they play a role in splicing of other viral transcripts including primary EBNA and Latent Membrane Protein (LMP) transcripts. Furthermore, it has been suggested that EBER1, 2 neutralize the transcription block normally induced by eIF-2^ref, mediate prevention of apoptosis^ref, suppress antiviral effects of IFN-a and -g and induce IL-10 production^ref. These effects may be mediated by the ability of EBER1, 2 to interact with and inhibit the function of the double stranded RNA-activated protein kinase PKR, a crucial mediator of interferon-induced antiviral effects. Recently an independent role in malignant transformation was suggested^ref.
• EBV nuclear antigen (EBNA)1 transcripts can be derived from 4 different promoters. During the initial stages of primary infection, (i.e. at the time of recircularization of the genome), transcription is initiated in BamHI-W (Wp)^ref; once host cell transformation is established, there is a switch towards the promoter in BamHI-C (Cp)^ref. Primary transcripts from these 2 promoters are very long (ca. 50–70 kb) and contain coding sequences not only for EBNA1, but also for the other EBNAs^ref. It is thought that these polycistronic transcripts are spliced, resulting in bi- or monocistronic transcripts, dependent on the needs of the virus at a certain time point. In latency types I and II where EBNA1 is the only EBNA expressed, Cp and Wp are silenced by methylation^ref and transcription is initiated from a promoter in BamHI-Q (Qp)^ref. Qp is considered to be the true latent promoter for EBNA1 transcription and is regulated in a cell cycle dependent manner involving EBNA1 autoregulation as well as by cytokine-mediated regulation^{ref1, ref2}. The fourth promoter is localized in BamHI-F (Fp) and is activated upon entry in the lytic cycle^ref. These individual EBNA1 promoters provide different levels of EBNA1 transcripts, depending on the host cell type and its activation state^ref. EBNA1 is a nuclear protein consisting of a basic amino terminus, a Gly–Ala repeat segment of variable length, another short basic domain including a nuclear localization sequence and a long hydrophobic C-terminal domain with DNA-binding and dimerization activity^{ref1, ref2}. Recently the crystal structure of the EBNA1 C-terminal dimer domain, bound to its cognate DNA sequence, was resolved revealing unexpected strong structural homology with the Bovine Papillomavirus E2 protein^ref. EBNA1 dimers bind to DNA by interacting with
• a partial palindrome sequence [TAGGATAGCATA-TGCTACCCAGATCCAG] that is present at 2 sites in the EBV genome^ref. There are 2 high-affinity sites: a set (family) of 20 tandem repeats of the cognate sequence (FR), and a combination of 2 sequences in dyad symmetry and 2 in tandem (DS). FR and DS elements are separated by a 1 kb intervening sequence. Together these form oriP, the origin of plasmid replication^{ref1, ref2}. Binding of EBNA1 to these sites results in looping-out of the 1 kb sequence between FR and DS, thus enabling the replication and persistence of the episome in cycling cells^ref; at the same time, EBNA1 is associated with host-cell chromosomes during mitosis which is important for segregation of episomes into progeny nuclei^{ref1, ref2, ref3}
• the third EBNA1 binding site is downstream of the latency I promoter in BamHI Q. EBNA1 binding to this site represses Qp activity, but this repression can be overcome by the presence of E2F and thus activity of Qp is cell-cycle dependent^{ref1, ref2}.
• EBNA1 furthermore enhances transcription from cellular^ref and at least 2 key latency-associated EBV promoters (Cp and LMP1p), which is correlated with its ability to link distant DNA sequences^{ref1, ref2} and binding to host-cell nucleosomal transcription and splicing factors, such as P32/Tap and EBp2^{ref1, ref2, ref3, ref4}.
In addition to its role(s) in maintenance of the viral episome and promoter activation, an oncogenic potential has been ascribed to this protein: mice carrying an EBNA1 transgene under control of the Ig heavy chain enhancer develop lymphomas^ref. Furthermore, the ability of EBNA1 to induce expression of the recombinase-activating genes RAG-1 and -2 is thought to lead to genetic instability^{ref1, ref2}. These observations may be relevant in the multistep oncogenesis as discussed above. EBNA1 is the only EBV-protein that is thought to be expressed in all latently EBV-infected cells. Although EBNA1 is a foreign protein to the host, EBV-infected cells expressing EBNA1 are not killed by CTLs. This is due to a inhibitory effect of the protein's Gly/Ala repeat (GAr) domain on proteasomal processing thereby preventing endogenous MHC class I-restricted presentation. This is likely to prevent peptide production from defective ribosomal products (DRiPs) of EBNA1 translation, a  key source of MHC class I presented peptides^ref. Although EBNA1 is barely recognized by CD8⁺ T-cells, CD4⁺ T-cells and antibodies reactive with EBNA1 are readily detected in most healthy virus carriers^{ref1, ref2}. The latter most likely arise from cross-priming by dendritic cells loaded with EBNA1 that is acquired via digestion of apoptosed EBV-positive cells^{ref1, ref2}.
• EBNA2 is encoded in the long primary transcripts initiated from Wp and Cp^ref and is the first EBV protein to be expressed after delivery of the viral genome to the nucleus. EBNA2 is a nuclear protein that in vitro specifically trans-activates cellular genes, such as the B-cell activation markers CD23 and CD21^ref and the c-fgr proto-oncogene^ref, and viral genes including LMP1^ref, LMP2^ref and the cis-acting element upstream of Cp^ref. The interaction of EBNA2 with its responsive elements is not a direct one but instead occurs via the RBP J-k protein^ref, resembling the signaling exerted by the Notch protein pathway^{ref1, ref2}. Together with EBNA5, EBNA2 is involved in G₀-G₁ transition^ref. Significant sequence diversity exists between the EBNA2 proteins of EBV types 1 and 2, which directly relates to functional differences such as in vitro transforming activity^ref. Although EBNA2 is essential for initial growth transformation of EBV-infected B-cells in vitro^ref, its expression may be less relevant for malignant growth in vivo because EBNA2 is not expressed in most EBV-associated tumors, except those in immunocompromized individuals. Even there EBNA2 expression seems to be limited and rather heterogeneous at the single cell level^ref.
• EBNA leader protein (EBNA-LP, EBNA5) is encoded in the leader of the Wp driven EBNA mRNAs^ref and is expressed simultaneously with EBNA2 in freshly infected B-cells^ref. The ORF is encoded by repeating exons in BamHI-Y and 2 short exons in BamHI-Y leading to the synthesis of multiple protein species ranging from 20 to 130 kDa^ref. Distinct functional domains on LP were recently mapped^ref. Whether the protein is actually synthesized, depends on the splicing of the mature mRNA. EBNA-LP function and phosphorylation appear to be cell cycle dependent^ref [110]. In vitro experiments have indicated that EBNA-LP is essential for transformation and directly or indirectly (via EBNA2) upregulates the expression of host factors required for B-cell growth^{ref1, ref2} [103 and 111]. Moreover, EBNA-LP was shown to bind p53 and pRb and cellular PKCs HA95 and HAX1, which regulate transcription by specific phosphorylation pathways resembling B-cell receptor mediated activation^{ref1, ref2, ref3, ref4} [103, 112, 113 and 114], leading to G₀–G₁ transition of resting B-cells. This is an important step in the early stages of EBV-mediated transformation.
• EBNA3A (EBNA3), EBNA3B (EBNA4) and EBNA3C (EBNA6), like EBNA2 are encoded in the long Wp and Cp driven primary transcripts^ref [64]. Mature mRNA encoding these 3 proteins are the least abundant of all EBNA mRNAs. EBNA3A, B and C are nuclear proteins of 140, 165 and 155 kDa, respectively, and localize into large nuclear clusters associated with the nuclear matrix, but excluding the nucleolus^{ref1, ref2} [115 and 116]. The EBV type 1 and 2 strains show a sequence identity of only 84, 80 and 72%, respectively, mainly due to variations in the C-terminal repeat regions, thus allowing strain typing. Reverse genetics have shown that EBNA3B is dispensible for B-cell growth transformation^ref [107]. On the other hand EBNA3B expression correlates with upregulation of CD40 and downregulation of CD77 in vitro^ref [117]. EBNA3A–C regulate the expression of certain cellular genes and bind to a variety of host proteins including different isoforms of the cellular transcription factor RBP J-k^{ref1, ref2} [118 and 119], thus modulating the EBNA2-driven upregulation of cellular and viral promoters^ref [120].
• EBNA3 / EBNA3A induces the nuclear accumulation of F538, a novel human uridine kinase/uracil phosphoribosyltransferase that is part of the ribonucleotide salvage pathway. Increased intranuclear levels of UK/UPRT may contribute to the metabolic build-up that is needed for blast transformation and rapid proliferation^ref
• EBNA3C upregulates CD21 expression in vitro^ref [121], augments the EBNA2-driven upregulation of LMP1 expression^ref [122] and downregulates Cp-promoter activity^ref [123]. EBNA3C resembles Adenovirus E1A and Papilloma virus E7 proteins by its ability to disturb cell cycle checkpoints and can override the G₂M checkpoint imposed by UV irradiation or genotoxic drugs such as etoposide and cisplatin, thus contributing to accumulation of DNA damage^{ref1, ref2, ref3} [124, 125 and 126]. Because the expression of EBNA3C in EBV-associated neoplasms in vivo has received little attention thusfar, its role in supporting tumor outgrowth remains to be determined.
• LMP1 mRNA originating from the BNLF1 ORF is a highly abundant viral transcript in most latently infected B-cell lines. The LMP1 protein is encoded by 3 exons and is an integral membrane protein with its hydrophilic (small) N- and (large) C-terminus positioned in the cytoplasm flanking 6 short hydrophobic membrane-spanning -helices linked by three short reverse turns that are predicted to loop-out on the extracellular side of the membrane^ref [127]. LMP1 forms patches in the cell membrane^ref [128] and associates with various intracellular membrane vesicles^ref [129] possibly mediated via its association with the intermediate filament protein vimentin^ref [130]. Part of the LMP1 may be secreted from EBV-infected cells in the form of MHC-II containing secretory vesicles, called exosomes^ref [131]. The domains responsible for patch formation comprise the highly conserved N-terminus and first 2 membrane spanning domains^ref [132]. Newly synthesized LMP1 remains detergent soluble for about 2 h, before it becomes phosphorylated and relocates to detergent insoluble cytoskeleton-associated intracellular structures^{ref1, ref2} [128 and 130]. > 50% of LMP1 is located intracellularly and even in clonal and synchronized EBV⁺ B-cell populations LMP1 expression is highly heterogeneous from cell to cell^ref [133]. In some viral strains induction of the viral lytic cycle leads to a boost of LMP1 expression, which, in part, may be derived from a second start site on the mRNA, resulting in a 45 kDa truncated form, largely lacking the transmembrane domains and devoid of most characteristic functions of LMP1^ref [134]. When expressed at low level LMP1 has clear growth enhancing and oncogenic effects^ref [135], but when expressed at high levels LMP1 causes general cytostasis^ref [136]. The cytostatic activity of LMP1 is linked to the first 2 transmembrane domains of LMP1^ref [137], which coincide with the T-cell inhibitory domain LALLFWL located in the first transmembrane helix at AA34–40^ref [131]. The balanced expression of LMP1 and its relative positioning on intracellular and plasma membranes in EBV transformed cells may prove to be relevant for the cell's growth potential. The level of LMP1 expression in different EBV-infected cell lines in vitro shows considerable variation similar to the LMP1 expression in EBV carrying tumor cells in vivo^{ref1, ref2, ref3, ref4} [106, 138, 139 and 140]. The expression of high levels of LMP1 in vivo has been linked to a worse prognosis^ref [141]. LMP1 is considered to be the major EBV-encoded transforming gene for several reasons^ref [39]: transfection of LMP1 has growth transforming effects in rodent fibroblasts^ref [142] and induces many of the changes that are usually associated with primary EBV-infection and transformation of B-cells^ref [143]. Moreover, LMP1 inhibits apoptosis in B-cells by inducing bcl-2^ref [144] and A20^ref [145]. In addition, LMP1 was shown to upregulate the cytokine IL-10 that stimulates B-cell proliferation and inhibits local immune response^ref [146]. In addition, studies with LMP1 transgenic mice have demonstrated direct oncogenic potential for LMP1. Mice carrying an LMP1-transgene under control of the Ig heavy chain promoter and enhancer develop lymphomas^ref  [147]. Upregulation of A20 and Bcl-2 expression in these transgenic mice was observed, in line with the previously found in vitro effects of LMP1. Mice carrying the LMP1 transgene under control of the polyoma virus enhancer and promoter (which directs specific expression in epithelia) show hyperplasia of the epidermis^ref  [148]. It was suggested that LMP1 expression—probably in association with aberrant expression of other (host) genes—may predispose to carcinogenesis. LMP1 was shown to interact with cellular proteins that are mediators of cytoplasmic signaling from the family of TNFR^ref [149]. This interaction is crucial for LMP1 to stimulate cell proliferation, both in B-cells and epithelial cells^{ref1, ref2} [150 and 151]. Thus, LMP-1 mimics the CD40–CD40L mediated NF-kB signal transduction pathway by interacting with specific members of the TRAF's (esp. TRAF1 and TRAF3) and TRADD^{ref1, ref2} [152 and 153]. LMP1 and CD40 co-localize in lipid rafts, but LMP1-associated TRAF-mediated signaling seems to be more efficient^ref [154]. The TRAF1, 3 interaction leads to NF-kB activation resulting in morphological changes and enhanced expression of B-cell activation markers including CD23, CD39, CD40, CD44, MHC class II and the cellular adhesion molecules LFA-1 and ICAM-1 [155]. LMP1 also induces enhanced signaling through the JAK–STAT pathway via binding to JAK3 kinase and ERK MAPK signaling, which involves TRAF2 as signal mediator^ref [156]. All these effects are mediated by at least three distinct signal activating domains (CTAR1-3) located in the cytoplasmic C-terminal half of LMP1^ref [153]. The hydrophilic N-terminus and first hydrophobic membrane-anchoring domain of LMP1 are essential for LMP1 function^ref [132] and mediate LMP1 association with distinct glycosphingolipid-rich raft domains in various cell membrane compartments^{ref1, ref2, ref3} [154, 157 and 158]. LMP1 has distinct effects on cytokine and cytokine receptor synthesis^ref [159], that may affect angiogenesis^ref [160] and inflammatory responses at the single tumor cell level in vivo^ref [161], thus contributing to tumor growth and immune escape^{ref1, ref2, ref3, ref4} [159, 161, 162 and 163].
• LMP2A and LMP2B are encoded by spliced mRNAs transcribed from the region spanning the terminal repeats of the EBV genome. Therefore, LMP2A, B mRNA is only transcribed once the circular viral episome is formed^ref  [164]. They have separate promoters resulting in unique first exons with LMP2B missing most of the N-terminal domain; the remaining exons are common for LMP2A and B. The LMP2 proteins both have 12 putative transmembrane domains and a hydrophilic C-terminal domain and colocalize with LMP1 in raft domains^ref [158]. It was shown that the LMP2A 119 aa N-terminal cytoplasmic domain, which is lacking in LMP2B, interacts via multiple phosphotyrosines arranged in ITAM- and SH2-protein binding motifs with the tyrosine kinases Lyn and Syk^ref [165]. This interaction diminishes Syk and Lyn from binding to the cytoplasmic B-cell receptor signalling domains, thus preventing antigen-receptor mediated activation of EBV-infected B-cells, that would otherwise result in induction of the lytic cycle. LMP2 is not required for B-cell transformation and has no growth altering effects when expressed in differentiating epithelia^{ref1, ref2} [39 and 166]. Studies with LMP2A transgenic mice have shown that LMP2A—although not oncogenic in these mice—can provide an survival signal to B-cells even when these do not express a B-cell receptor^{ref1, ref2} [167 and 168]. It was suggested that this signal, in combination with the B-cell activation-preventing activity of LMP2A, is important for persistence of EBV in the host^ref [165]. This is further supported by recent data showing a well regulated co-expression of LMP1 and LMP2, together with EBNA1, in circulating EBV carrying memory B-cells travelling through lymphoid follicles of the tonsil in vivo. This tightly regulated expression of LMP1 and LMP2 appears to be important for maintaining long-term persistence, by providing temporal growth and survival signals during passage of these cells through lymphoid organs^{ref1, ref2, ref3} [169, 170 and 171].
• rightward transcripts of the BamHI-A region of the viral genome (BARTs) can be detected in all types of EBV latency, including EBV-infected peripheral blood B-cells and are expressed at particularly high levels in nasopharyngeal carcinomas^{ref1, ref2, ref3, ref4, ref5}[172, 173, 174, 175 and 176]. Interestingly, these transcripts are co-terminal and harbour the BARF0 open reading frame (ORF) at their 3?-ends^{ref1, ref2, ref3} [177, 178 and 179]. This ORF encodes a putative protein of 174 amino acids. By means of alternative splicing, the BARF0 ORF may be extended with 105 additional amino acids. The alternative ORF thus generated is called RK-BARF0^ref [180]. Whether (RK-)BARF0 protein is expressed in vivo is at present uncertain. The presence of a polyadenylation signal 5' to the stopcodon renders actual translation unlikely^{ref1, ref2} [177 and 179], but anti-BARF0 antibody [180] and cytotoxic T lymphocyte (CTL)^ref  [181] responses detected in patients suggest that synthesis of this protein does take place. However, a publication from the same group indicated that alternative splicing may remove the actual BARF0 coding sequences from BART's and suggested that the original antibody used to detect the (RK-)BARF0 proteins also detects a cellular component of similar molecular weight^ref [182]. Work from our own group, using newly developed monoclonal antibodies reactive in vitro with various native and denatured recombinant forms of the (RK-)BARF0 protein, did not result in any evidence for (RK-)BARF0 protein expression in vivo in multiple EBV⁺ tumor and EBV-transformed cell line specimens, despite the clear detection of abundant spliced and non-spliced RNA transcripts encoding the putative (RK-)BARF0 protein species. In addition, we could not reproduce the published antibody detection results using purified (RK-)BARF0 protein or defined peptide domains thereof as antigen^ref [183]. Therefore, there remains considerable dispute on a possible role for (RK-)BARF0 protein EBV-infection in vivo. Recently, in vitro functional studies were initiated using heterologous expressed recombinant (RK-)BARF0 protein. One of these studies showed a nuclear localization of the protein^ref [184]. Transfected RK-BARF0 was shown to induce the expression of LMP1, a mechanism dependent on the apparent interaction between RK-BARF0 and Notch^ref [185]. It was hypothesized that this mechanism guarantees expression of LMP1 in EBNA2-lacking latency types. The RK-BARF0 protein, however, appears to turn over rapidly with Notch^ref [185], which could be the reason why the protein could not be detected in EBV-associated diseases^ref [183]. At least 2 other putative ORFs were demonstrated in BARTs, called RPMS1 and RPMS2 / A73^{ref1, ref2, ref3} [173, 186 and 187]. RPMS1 encodes a putative nuclear protein^{ref1, ref2} [173 and 186] that is partially homologous to EBNA2, in particular the WWP containing domain that interacts with RBP J-k. It is tempting to speculate that RPMS1 acts as a substitute for EBNA2, for example in latency types I and II. However, recent data point to a negative influence of the putative RPMS1 protein on EBNA2 and Notch signalling^ref [188]. Although transcription of RPMS1 in latently infected B-cells from healthy donors was found^ref [173], neither transcription of RPMS1 in patient samples nor expression of RPMS1 protein have been reported. Even less is known about potential expression and function of the A73 protein which has been suggested to modulate integrin signalling pathways^ref [186]. Finally, a role for BARTs in transcriptional control was suggested, by virtue of their anti-sense orientation relative to several important early–late leftward gene transcripts, such as BALF5 (DNA-polymerase), BALF4 (the nuclear transport membrane protein gp125) and BILF1, a putative cytokine receptor^{ref1, ref2, ref3, ref4, ref5} [39, 175, 176, 177 and 186]. Thus, BARTs could play a role in maintenance of latency. BARF1 is a rightward transcript from the BamHI-A region that has its own promoter. Originally recognized as an early lytic phase transcript^ref [189]. However, recent data indicate that BARF1 is preferentially expressed in EBV carrying epithelial tumors but not in lymphoid tumors^{ref1, ref2, ref3, ref4, ref5} [50, 54, 190, 191 and 192]. This classifies the BARF1 gene as an interesting and useful carcinoma-specific marker. The BARF1 protein has transforming properties both when expressed in B-cells^ref [193] and in epithelial cells^ref [194]. The transforming domain of BARF1 may be located in the N-terminus and mediates Bcl2 upregulation^ref [195]. The extracellular domain of BARF1 may be selectively cleaved off and secreted from the cell and is considered to be a functional, soluble homolog of the human colony stimulating factor 1 (CSF-1) receptor^ref [196]. As such, BARF1 is hypothesized to play a role in local immune evasion by absorbing CSF-1 that is necessary for an effective immune response. Thus, BARF1 constitutes a protein with pleiotropic function, involved both in oncogenesis and immune escape with a possible significant role in EBV-associated carcinomas, like NPC and GC.
Expression patterns of latent genes :
• latency I (first detected in Burkitt's lymphoma (BL)) : EBNA1, BARTs and EBERs (10⁶ copies per cells)
• latency II (first detected in nasopharyngeal carcinoma (NPC) but this also proved to be the prevailing expression pattern in most other EBV-positive tumors that occur in the immunocompetent host, such as Hodgkin's lymphoma (HL) and T- and B-cell NHLs) : EBERs, BARTs, EBNA1 + LMP1 + LMP2A + LMP2B
• latency III (lymphoblastoid cell lines (LCLs) in vitro) : EBNA1 + EBNA2 + EBNA3A + EBNA3C +  LP + LMP1 + LMP2. However, in vivo, expression of the complete set of latent genes is only found in absence of a fully functional immune response, i.e. in lymphomas of AIDS patients and of transplant recipients. Under these circumstances individual tumor cells may display a considerable degree of expression heterogeneity, overall resembling latency-III^{ref1, ref2} [106 and 201]
• several other gene expression patterns are recognized that cannot be grouped with the known latency patterns^ref [191]
Overview of EBV protein expression (for which suitable antibodies are available) in EBV-associated diseases

++, Positivity in >75% of neoplastic cells in all tested tumors; +, positivity in 10–50% of neoplastic cells in most of the cases; ±, positivity in <5% of neoplastic cells in part of the cases. Abbreviations: (see also Chapter 1) ePTCL, extranodal peripheral T-cell lymphoma; GC, gastric carcinoma; nd, not determined; ±, weak positivity in part of the cases; immunohistochemistry in these cases shows signals in small percentage of neoplastic and reactive cells; in isolated cases, weak transcription is found, although not confirmed by immunohistochemistry; therefore, these signals are ascribed to EBV-infected reactive cells.

• lytic phase : during the lytic replication phase of the EBV life cycle, many more viral genes are expressed which encode proteins involved in viral DNA replication and viral particle synthesis
• kinases expressed only during the lytic form of infection
• thymidine kinase
• BGLF4 phosphorylates the viral early antigen EA-D^ref
• immediate early (IE) gene products : genes which are expressed immediately upon induction of the lytic cycle, independently of new protein synthesis, and which encode transcription factors that are crucial for activation (switching-on) of lytic phase genes; essentially considered to form the switch between latent and lytic cycle. However, IE gene expression cannot be considered synonymous to full viral lytic replication and on the other hand some late gene products may be expressed without concomitant IE gene expression^{ref1, ref2} [39 and 54].
• BZLF1 / Zebra (Z) : the best studied IE gene product; a powerful trans-activator of early EBV gene expression^ref [39]. The Zebra protein consists of a trans-activating domain, a basic domain that has homology to a conserved region of the c-jun/c-fos family of transcription factors^ref [197], and a domain that enables Zebra to interact with p53^ref [198]. Overexpression of p53 and g-irradiation have been shown to induce Zebra expression in a NPC-derived EBV carrying epithelial cell line leading to the (partial) expression of lytic cycle genes^ref [199]. In most EBV-associated malignancies sporadic tumor cells show detectable Zebra protein expression, probably reflecting partial (abortive) activation of the lytic cycle, because true late gene transcription is mostly absent^{ref1, ref2, ref3, ref4} [54, 200, 201 and 202]. Expression of Zebra may interfere with IFN signalling, by decreasing IFN-receptor expression and preventing IFN-induced STAT1 tyrosine phosphorylation together, resulting in abrogation of IFN-induced MHC-II upregulation and thereby contributing to immune escape^ref [203]. BZLF1 converts the virus from the latent to the lytic form of infection even when the viral genome is highly methylated. Methylation of CpG motifs in Z-responsive elements of the viral BRLF1 immediate-early promoter enhances Z binding to, and activation of, this promoter. Demethylation of the viral genome impairs Z activation of lytic viral genes. Z is the first transcription factor that preferentially binds to, and activates, a methylated promoter^ref.
• BRLF1, BRRF1 (R) and the BI-LF4 transactivator genes^{ref1, ref2} [39 and 204], have been studied only little in relation to EBV+ tumor development and will not be discussed here.
BZLF1 and BRLF1 both encode transcriptional activators, and together these proteins induce transcription of the entire lytic viral gene program. In latently infected B cells, the BZLF1 (Zp) and BRLF1 (Rp) promoters are inactive. However, ligation of the B-cell receptor^ref, phorbol ester treatment^ref, calcium ionophores^ref, TGF-ß₁^ref, demethylating agents^ref, and agents which induce histone acetylation^{ref1, ref2, ref3} are known to activate expression of the BZLF1 and BRLF1 IE promoters in at least a portion of EBV⁺ B-cell lines. In the case of the BZLF1 IE promoter (Zp), the two promoter elements which appear to be essential for stimulation by most, if not all, of these inducing factors are termed the ZI and ZII motifs^ref. Several of the ZI motifs are bound by the MEF2D cellular transcription factor, as well as by Sp1/Sp3^{ref1, ref2}, and MEF2D has been shown to be an important regulator of EBV infection in host cells^{ref1, ref2}. The ZII motif is a CRE site which is bound by CREB, ATF-1, c-Jun, and ATF-2^{ref1, ref2}. The ability of both gemcitabine and doxorubicin to activate BZLF1 transcription in EBV-negative cells requires both the ZI and ZII binding motifs of the BZLF1 promoter. Gemcitabine and doxorubicin also activated the BRLF1 IE promoter in EBV-negative cells. This effect was shown to require the presence of 2 EGR-1 (Zif268) binding sites in the promoter which were previously shown to be important for phorbol ester-induced activation of the promoter^ref.
• early gene products : genes of which the expression is not affected by inhibition of viral DNA synthesis including series of enzymes influencing the host cell nucleotide metabolism and DNA synthesis; early lytic genes are barely expressed in EBV-associated malignancies and are not considered to contribute to the oncogenic process, with the possible exception of BHRF1. However, their expression can be induced by chemical treatment, irradiation or membrane receptor triggering of latently infected cells, which is mediated by prior expression of the EBV IE transactivator protein Zebra^ref [39]. Enzymes included among the early lytic genes are potential targets for antiviral drugs which may lead to applications for future tumor therapy^{ref1, ref2, ref3} [199, 205 and 206].
• BHRF1 mRNAs are abundantly expressed from their own promoter in BamHI-H (Hp) during early lytic infection^ref [39]; the protein can also be detected then and relates to the 17 kDa EA-R antigen^ref [207]. However, the protein is not detectable in most latently infected cell lines^ref [208] and transcripts of BHRF1 in these cells include leader sequences found in Cp/Wp EBNA transcripts^ref [209]. In vivo, BHRF1 transcripts are predominantly found in EBV-associated B-cell lymphomas^ref [210], although expression at the protein level apparently does not occur significantly^{ref1, ref2} [211 and 212]. Low level BHRF1 transcription occasionally can be detected in Hodgkin's lymphoma (HL) and in T-cell NHLs and even in nasopharyngeal carcinoma (NPC) and BHRF1 mRNA and protein expression is particularly abundant in oral hairy leukoplakia (OHL)^{ref1, ref2} [54 and 212]. Interestingly, BHRF1 shows structural and functional homology to the host-encoded apoptosis inhibitor Bcl-2^ref [213] and is highly conserved among gamma herpesviruses, suggesting an evolutionary conserved important role for BHRF1-protein in vivo^{ref1, ref2} [214 and 215]. It is thought that BHRF1 mediated apoptosis-inhibition contributes to prevent early lysis of productively infected cells by cytotoxic T-cells thus prolonging virus production time. A deregulated expression of BHRF1 in EBV tumor cells may similarly contribute to enhanced tumor cell survival [216].
• late gene products : genes of which the expression relies on new linear genomic templates and whose expression is blocked by inhibition of lytic (linear) viral DNA synthesis, including
• most of the virion structural proteins. Most late genes that can be identified directly or based on their homology with other herpesvirus genes encode structural proteins. Amongst these are :
• VCA p18 (BFRF3) the small capsid protein
• VCA-p40 (BdRF1) the scaffold protein^ref [217] 
• Gp125 (BALF4) the nuclear membrane protein
They are strongly immunogenic in humans and serve as targets for serodiagnosis, because virtually all EBV carriers develop antibodies to these proteins^{ref1, ref2} [92 and 218].
BLLF1 encodes the major virion envelope glycoprotein Gp350/220 that mediates virion binding to CD21 / CR2 / EBVR and is the major target of the neutralizing antibody response^ref [219].
• non-structural genes :
• viral IL-10 (BCRF1)^{ref1, ref2} is of particular interest because of its close homology with human IL-10 (hIL10). The structural homology consists of nearly 90% colinear amino acid sequence identity^ref [220] and BCRF1 is also functionally homologous to hIL10^{ref1, ref2} [221 and 222]. They share the ability to modulate local immune responses, to inhibit the function of macrophages and NK cells and the production of interferon-. Similar to the BHRF1 encoded viral bcl-2 homologue, viral IL-10 probably serves to enhance survival of virus producing cells in a hostile inflammatory environment. Both genes were probably acquired by EBV as a consequence of close co-evolution with its human host within cells of the immune system providing a selective survival advantage. In vivo, vIL10 (BCRF1) expression is mainly restricted to OHL in AIDS-patients^ref [54]. OHL is a lesion with a unique combination of latent and lytic EBV gene expression and forms the only proliferative disorder associated with lytic EBV replication^ref [151]. In contrast, hIL10 expression seems to predominate in most EBV-associated malignancies, probably directly linked to the expression of LMP1^ref [161]. Overall IL-10 expression is elevated in most EBV-associated diseases and can be detected both in affected tissues and in serum and is shown to correlate with a bad prognosis^{ref1, ref2, ref3} [223, 224 and 225].

Transmission : direct or indirect through oropharynx ("kissing disease"), blood or vehicles. Under normal circumstances, EBV-infection is restricted to humans, although some types of monkeys can be infected experimentally^ref. EBV enters epithelial cells in the oropharyngeal mucosa through 3 CD21-independent pathways:

• by direct cell-to-cell contact of apical cell membranes with EBV-infected lymphocytes
• by entry of cell-free virions through basolateral membranes, mediated in part through an interaction between b₁or a₅b₁ integrins and the EBV BMRF-2 protein
• after initial infection, by virus spread directly across lateral membranes to adjacent epithelial cells.

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-infection^ref [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 saliva^ref [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 C3d^{ref1, ref2} [227 and 228]. After CD21 binding the viral envelope fuses with the host cell membrane and triggers host cell activation^{ref1, 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 transcription^ref [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 used^{ref1, 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 EBV^{ref1, ref2, ref3} [232, 233 and 234]. In the first stages after recircularization of the genome, the full spectrum of latent proteins is expressed^ref [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-blasts^{ref1, 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 persistence^{ref1, 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 syndrome^ref [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 LMP2^ref [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 cells^{ref1, 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 driven^ref [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 virus^ref [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 EBV^{ref1, 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 vitro^ref [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 established^{ref1, ref2, ref3, ref4, ref5} [39, 246, 247, 248 and 249]. This silencing is accomplished by methylation of early latency promoters Cp and Wp^{ref1, ref2, ref3} [66, 250 and 251]. Because EBNA1 is indispensible for maintenance of the viral genome in the dividing host cells^{ref1, 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 presentation^ref [90]. This is considered to be an important mechanism by which EBV⁺ tumor cells escape CTL-mediated killing^ref [249]. Given the fact that memory CTLs reactive against most EBNAs remain clearly detectable for life^{ref1, 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 tonsil^{ref1, 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 oropharynx^ref [241]. It is thought that this switching process is influenced by signals that normally control B-cell behaviour, such as antigen-driven activation^{ref1, 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 tonsil^{ref1, 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 phenomenon^ref [254]. Local antigen triggering and CD40 activation may subsequently lead to virus production and transepithelial secretion^ref [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) complex^{ref1, 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 nodes^ref [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 functions^{ref1, 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 cells^{ref1, 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 disorder^ref [212]. Particularly strong expression of early BHRF1 transcripts is confined to OHL^{ref1, 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 transcript^ref [189]. Using a reversed northern blotting technique, the transcript was found to be preferentially expressed in epithelial but not in lymphoid cells^ref [190]. More recently, using NASBA, we found BARF1 transcription exclusively in NPCs and in OHL but not in lymphoid disorders^{ref1, ref2} ( Fig. 5) [50 and 54]. In addition to these studies, BARF1 transcripts were also detected in EBV-associated gastric carcinomas^ref [191]. Recently, expression of BARF1 at the protein level was detected in NPC biopsies by immunoblotting^ref [192]. These findings indicate that BARF1 expression is specific for EBV-associated epithelial malignancies. BARF1 has clear effects on epithelial cells in vitro^ref [194], in more than one aspect mimicking the role of LMP1 in B-cells^ref [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-B^ref [54] and the BFRF1 putative virion protein, remain to be explored^ref [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 HDs^ref [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 intact^ref [362]. This is also found for NPC and T/NK cell lymphomas^{ref1, 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 CTLs^{ref1, 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 cells^ref [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 EBV^ref [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 PCNA^{ref1, 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 HD^ref [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-10^{ref1, 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 origin^ref [161]. Upregulation of human IL-10 upon EBV-infection in vitro has been demonstrated^ref [365], and, interestingly, elevated human IL-10 expression levels have been detected in the H-RS cells of EBV-positive HDs^{ref1, 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 expression^{ref1, ref2} [96 and 444]. Moreover, alternative splicing of c-fgr transcripts was reported in EBV-positive cell lines^ref [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 profiling^ref. EBV-induced gene 3 (EBI3) is expressed in DCs and is part of the cytokine IL-27^ref that controls T_h 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 T_h cell development^ref. 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 EBI3^ref
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 vivo^ref.

Symptoms & signs :

• largely subclinical in early childhood : latency II and III expression patterns in tonsils of healthy asymptomatic healthy carriers
• lymphoid diseases :
• infectious mononucleosis (IM) (18%; once termed Drusenfieber, i.e. Pfeiffer's glandular fever) after 30-60 days incubation (10-15 days in babies) : colonization of Waldeyer's ring => fever (66-76%, only in first 10 days), cough (14%), pharyngotonsillitis (1.6%; "kissing tonsils" : if severe bacterial overinfection and oedema => cortisone therapy); anterior and posterior laterocervical, inguinal and axillary lymphadenomegaly (5-94%, for 2-3 weeks), mild hepatomegaly (12%; increase in SGPT up to 100÷200 U/L at 37°C), eyelid edema (5.3%), infantile hepatitis syndrome (0.5%), jaundice (9%), splenomegaly (52%) due to red pulp hyperplasia (sometimes spleen upper pole may enlarge from the 7^th÷8^th intercostal space down to the left iliac cavity : rare splenic infarction and splenic rupture; avoid splenic injuries during recovery !), maculopapular exanthema (8-10%), mild sinusal tachycardia (1.6%), hyperhemic tonsils with no exudate, gross hematuria (0.5%), pericarditis and interstitial pneumonia. Genital ulcerations can be the initial manifestation of EBV in adolescents who are neither sexually active nor immunocompromised practicing cunnilingus^ref. IM can be considered the clinically manifest form of a primary EBV-infection. Its diagnosis relies in the detection of atypical lymphoid cells in the peripheral blood, the occurrence of so-called heterophile antibodies and EBV-seroconversion^ref [276]. IM is a benign disorder with expansion of the paracortex of lymphoid tissues. The proliferating cell populations are EBV-infected polyclonal B blasts^ref [277], accompanied by the growth of activated T-cells^ref [278]. Morphologically, the EBV-infected cells range from large immunoblasts, including H-RS like cells, to small lymphoid cells. In addition to latently infected cells, a small number of EBV-positive cells expressing lytic cycle antigens such as BZLF1 can be detected in tissues from IM patients. These are often found adjacent to crypt epithelium and are frequently positive for plasma cell markers^{ref1, ref2, ref3} [252, 253 and 279]. It is thought that EBV can replicate in these plasmacytic cells, thus generating a cellular source of infectious virus in the saliva of IM patients^{ref1, ref2} [241 and 253]. Further EBV gene expression analysis has shown a type III EBV latency pattern in IM [235], although the expression at the single cell level is more heterogeneous. Most cells are EBER positive but do not express EBNA2 or LMP1 (type I latency), whereas some large immunoblasts express LMP1 in the absence of EBNA2 (type II latency) and many small lymphocytes express EBNA2 but not LMP1^{ref1, ref2} [237 and 279].

Laboratory examinations : leukocytosis (> 4,500÷10,000 / mL) with relative reactive lymphocytosis due to proliferation of Downey cells / atypical lymphocytes (i.e. CD8⁺ T_s cells) :
• type I cells : kidney-shaped or lobulated nucleus with vacuolated, basophilic foamy cytoplasm
• type II cells contain plasmacytoid nuclei with less vacuolated and basophilic cytoplasm
• type III cells have a finer chromatin pattern and 1-2 nucleoli.
..., decrease in HCT, HGB and MCV (differential diagnosis with massive hemolysis, internal or external hemorrhages, hemoglobinopathies)
Prognosis : self-limiting within 2-4 weeks.
Complications :
• acute lymphadenitis (1.0%)
• subacute necrotizing lymphadenitis (0.5%)
• facial neuritis (1.0%)
• acute aplastic anemia (0.5%)
• viral myocarditis (2.6%)
• EBV-associated hemophagocytic syndrome (EBV-AHS) (1.6%)
• post-infective meningoencephalitis (PIE : < 1%). EBV CNS infections are divided into 2 groups :
• CNS syndromes associated with primary EBV or reactivated infection : diverse CNS syndromes can occur
• acute viral encephalitis (2.6%) : convulsion (1.6%)
• acute cerebellar ataxia
• acute disseminated encephalomyelitis (ADEM) (sometimes positive PCR from CSF)
• myelitis
• meningitis (sometimes positive PCR from CSF)
• CNS syndromes associated with chronic EBV infection
• encephalitis
• relapsing ADEM
• X-linked lymphoproliferative syndrome (X-LPS) / fatal mononucleosis : caused by hereditary mutations in the gene encoding the signalling lymphocyte activation molecule (SLAM)-associated protein (SAP) on position q25 of the X-chromosome^{ref1, ref2} [280 and 281]. The self-ligand SLAM protein is present on the surface of both B and T-cells. When interactions occur between SLAM molecules on the interface between T- and B-cells, signal transduction pathways are initiated. The T-cell protein SAP binds to SLAM and as such acts as a negative regulator^{ref1, ref2} [282 and 283]. Individuals that have inherited this trait are usually asymptomatic, but upon primary EBV-infection their immune response becomes overreactive because of the non-functional SAP protein. This usually results in a fulminant IM accompanied by a EBV-associated hemophagocytic syndrome (EBV-AHS) by which liver and bone-marrow are destroyed^ref [284]. The only curative treatment for this syndrome is allogeneic bone-marrow transplantation^ref [285]. However, patients who do survive the infection are prone to developing lymphomas later in life^ref [286].
• EBV-mild acquired immune deficiency syndrome (EBV-MAIDS) in postsurgical sinusitis

Therapy : periodic intramuscular IVIg
• autoimmunity may cause :
• autoimmune hemolytic anemia
• autoimmune thrombocytopenic purpura (ATP) (5.8%)
• autoimmune granulocytopenia
• Guillain-Barré syndrome
• type IA insulin-dependent diabetes mellitus
• polydermatomyositis
• rheumatoid arthritis (1.0% : RF in < 5%)
• chronic fatigue syndrome (CFS) : inability to demonstrate a role for reactivation of EBV in CFS, even in selected patients with high titers of antibody to VCA and EA of EBV
• systemic lupus erythematosus (SLE) (0.5%)
• Kawasaki disease (6.3%)
• immunoproliferative syndrome / lymphoproliferative disease (LPD) (latency III expression pattern) in stem cell and organ transplant recipients (Cohen, 2000) : the present view that lymphomas have to be considered neoplastic counterparts of reactions normally occurring in lymphoid tissues after antigenic stimulation, is helpful in understanding their pathogenesis. Lymphomagenesis is considered to be a multistep process^ref, during which an accumulation of genetic changes takes place. Lately, numerous studies have identified cytogenetic abnormalities that are characteristic of specific non-Hodgkin lymphomas (NHLs). These are frequently translocations involving the antigen receptor loci. Since transcriptional activity of the antigen receptor loci is inherent to the function of lymphoid cells, translocations involving these loci usually result in overexpression of an oncogene under the control of the antigen receptor expression regulation. A characteristic example is the t(14; 18) in follicular lymphomas, which results in overexpression of the apoptosis inhibitor bcl-2 under the control of the Ig heavy chain promoter^{ref1, ref2}. The finding that cells containing t(14; 18) can be detected in the peripheral blood of healthy individuals^ref indicates that this translocation may be an early event in the genesis of follicular lymphoma. Another translocation involving the Ig heavy chain locus is t(8; 14) which occurs in many of the Burkitt's lymphomas (BL). This translocation results in overexpression of the transcription factor c-myc under the control of the Ig heavy chain promoter^{ref1, ref2}. The difference in growth rate between follicular lymphomas and BL is clearly the consequence of the different genes involved in their respective genetic alterations. The t(14;18) involving bcl-2 results in an extended lifespan of the affected cells, allowing the accumulation of more genetic hits. By contrast, genetic changes resulting in overexpression of a transcription factor usually have a direct influence on proliferation and, therefore, are usually associated with rapidly growing lymphomas, as is the case with c-myc in BL. Besides cytogenetic abnormalities, chronic antigenic stimulation is thought to play an important role in lymphomagenesis. The presence of antigens not only stimulates proliferation (resulting in ‘fixation’ of genetic aberrations throughout the next cell generations) but also induces the process of Ig rearrangement, and thus increases the possibility of more aberrant rearrangements to take place^ref. This can also be illustrated from the c-myc translocations in BL. Recent findings show that all cases of BL harbor somatically mutated V region genes and are as such derived from B-cells that took part in a germinal center reaction^ref. It was proposed that most, if not all, c-myc/Ig translocations in endemic BL happen in a mutating germinal center B-cell and result from the process of hypermutation as such. In sporadic BL, c-myc translocations are usually targeted to the class-switch region of the Ig gene, and thus probably relate to class switch recombination that also take place in the germinal center^{ref1Lenoir GM, 173-206, ref2}. Some lymphoma subtypes are clearly related to the presence of a certain antigen. For example, gastric lymphomas are related to infection with Helicobacter pylori (H. pylori)^ref, and certain cutaneous B-cell lymphomas are associated with Borrelia burgdorferi infection^ref. In AIDS-related non-Hodgkin Lymphoma (ARNHL) similar chronic antigen exposure may be responsible for tumor outgrowth, but the triggering antigen remains elusive^ref. For the endemic form of BL, the antigens providing chronic stimulation are probably Epstein–Barr virus (EBV) and malaria^ref. The latter provides a strong and acute B-cell activation stimulus mediated by repetitive epitopes on the parasite surface, whereas the action of EBV in BL may be 2-fold: it functions as antigen and as a transforming agent. The presence of somatic hypermutations in the variable region of the Ig heavy chain in BL cells strengthens the hypothesis that exposure to antigens may be relevant for the pathogenesis of BL^{ref1Lenoir GM, 173-206, ref2}. Furthermore, the presence of Ig-gene hypermutations in the Reed–Sternberg cells of Hodgkin's disease^ref and the expression of functional differentiation markers such as granzyme-B and TIA-1 in all EBV-associated T-/NK-cell NHL's^ref also suggest that these tumors are initially driven by antigen stimulation. Similar to the situation in endemic BL, the presence of EBV genome may provide immortalizing functions thus contributing to the multistep oncogenic process^{ref1, ref2}.
• autoimmune lymphoproliferative syndrome (ALPS)
• Kikuchi-Fujimoto disease (KFD) / subacute or histiocytic necrotizing lymphadenitis (HNL) ?
• EBV-associated T/NK-cell LPD : target cell specificity, defects in host immune responses, and strain differences of EBV may account for ectopic EBV infections and for the unique clinical presentations characteristic of each illness^ref.
• chronic infectious mononucleosis (CIM) / chronic Epstein-Barr virus infection (CEBV) / chronic active EBV infection (CAEBV) in apparently immunocompetent hosts (extraordinary event after acute disease)

Symptoms & signs : chronic or recurrent infectious mononucleosis-like symptoms (intermittent fever, weight loss and liver abnormalities, rarely multiple nodular coagulation necrosis in the liver) persisting over a long time and by an unusual pattern of anti-EBV antibodies^{Rickinson AB, 13-14}. Patients with this disease have no evidence of any prior immunologic abnormalities or of any other recent infection that might explain their condition^{Rickinson AB, 13-14, ref2}. CAEBV is a disease with a high mortality and high morbidity with life-threatening complications, such as virus-associated hemophagocytic syndrome, interstitial pneumonia, lymphoma, coronary artery aneurysms, and central nervous system involvement^{ref1, ref2, ref3, ref4, ref5}. The 3 main criteria of CAEBV infection are^ref :
• (1) severe illness lasting > 6 months that began as a primary EBV infection and that was associated with grossly abnormal EBV antibody titers, antiviral capsid antigens (VCA) IgG > 5120, anti-early antigens (EA) IgG > 640, or anti-EB nuclear antigens (EBNA) < 2;
• (2) histologic evidence of major organ involvement such as interstitial pneumonia, hypoplasia of some bone marrow elements, uveitis, lymphadenitis, persistent hepatitis or splenomegaly; and
• (3) increased quantities of EBV in affected tissues.
Subsequently, Okano and his colleagues^ref proposed similar criteria to diagnose severe CAEBV infection.
Importantly, many cases have been reported that do not satisfy the criteria described above. Some patients lack abnormal patterns of EBV-related antibodies, whereas other patients lack major organ involvement and have only skin symptoms, such as hypersensitivity to mosquito bites (HMB) or hydroa vacciniforme-like eruptions^{ref1, ref2}. On the other hand, patients with CAEBV infection had extremely high viral loads, as assessed by qPCR^{ref1, ref2}. Clonal expansion of EBV-infected T or natural killer (NK) cells could be associated with CAEBV infection^{ref1, ref2, ref3, ref4, ref5, ref, ref7}. Detailed clinical features of some patients have been described^{ref1, Morita M, 1485-1488, ref3}. An accumulating body of evidence suggests that clonal expansion of EBV-infected T or NK cells is associated with CAEBV infection^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}. In healthy carriers, EBV exists latently in resting memory B cells^ref. It is unclear whether the invasion of blood cells other than B cells causes CAEBV infection or the invasion is an ordinary event, but the host's immunologic abnormalities allow the expansion of these cells. Recently, a defect in the SAP/SH2D1A gene was implicated in XLP, a fatal lymphoproliferative disorder^{ref1, ref2, ref3}. Patients with XLP are exclusively boys in whom primary infection with EBV causes lymphoproliferation and severe hepatitis, mimicking CAEBV infection^{ref1, ref2}. Therefore, we examined the SAP/SH2D1A gene in all the male subjects enrolled in our study, but did not find any abnormalities. However, it is possible that a defect in another gene essential for regulating lymphocyte activation and proliferation may be a cause of CAEBV infection. Such a defect or single nucleotide polymorphism might influence the function of virus-specific or nonspecific lymphocytes and thereby allow the expansion of EBV-infected T or NK cells. It has been reported that EBV-infected T and NK cells express a limited range of viral antigens^ref. EBV-infected B cells express at least 9 antigens, some of which are antigenic enough to be presented to CTLs. This is called latent infection type 1. EBV-infected T and NK cells express only EBNA1 and LMP1; this is called latent infection type 2^ref. The expression of viral antigens was examined in some CAEBV patients, and they were type 2^{ref1, ref2}. Because these 2 proteins are less antigenic, infected cells can evade from immune surveillance by CTLs. Therefore, they may proliferate and cause chronic infection. Chromosomal abnormality occurred in the lymph nodes of patients with CAEBV^ref : 50% of the patients examined had chromosomal aberrations in their peripheral blood cells and 79% of patients showed monoclonality of EBV. Because there were no specific patterns of genetic aberration, and some patients displayed several different aberrations, the chromosomal abnormalities seen in patients with CAEBV infection might only reflect chromosomal fragility. However, clonality of the EBV genome and chromosomal aberrations generally indicate clonal expansion of EBV-infected cells. These results indicate that clonal expansion is a common feature of CAEBV infection, and this disease might therefore be considered lymphoproliferative rather than infectious^ref. In fact, patients with CAEBV infection frequently develop neoplasms such as malignant lymphoma. It would therefore be better to call the cases presented here "chronic EBV-associated lymphoproliferative disorders." Approximately 10 out of 30 patients with CAEBV infection did not meet the previously accepted definition of CAEBV infection^{ref1, ref2}. These patients had symptoms typical for CAEBV infection and had extremely high EBV loads in their peripheral blood. High titers of EBV-related antibody are not always necessary for the diagnosis of CAEBV infection. On the other hand, all the patients had > 102.5 copies/µg EBV DNA in PBMC. Furthermore, it is of note that the copy number of EBV DNA in PBMC decreased below 102.5 copies/µg DNA in all 7 patients who underwent hematopoietic stem cell transplantation. These results indicate that viral loads greater than102.5 copies/µg DNA can be used not only as diagnostic factors for CAEBV infection, but also as predictors of therapeutic efficacy. Previous reports show that patients with CAEBV infection had cell-free EBV DNA in plasma, although the origin of the viral DNA is unclear.37,38 Usually, the plasma of healthy individuals does not contain EBV DNA^{ref1, ref2}. Therefore, the presence of EBV DNA in plasma may have significance for the diagnosis of CAEBV infection. However, it should be borne in mind that the plasma of patients with CAEBV infection did not always test positive for EBV DNA. Indeed, in this study, EBV DNA was not detected in plasma from 6 patients, whereas they had large amounts of EBV DNA in their PBMC. PBMC, and not plasma, should be used for diagnosing and monitoring patients with CAEBV infection. It is unclear why some patients did not have cell-free EBV DNA. Patients with negative plasma results sometimes turned positive at a later visit. Cell-free viral DNA may fluctuate from day to day. The other possibility is that inhibitors in plasma might influence PCR reactions and cause false-negative results. In conclusion, patients with CAEBV infection were divisible into 2 subgroups: T-cell and NK-cell CAEBV infection. Each group had different clinical features and prognosis. A high titer of EBV-related antibodies is not always a prerequisite for the diagnosis of CAEBV infection. Viral load, detected by quantitative PCR in PBMC, was useful for disease diagnosis and as an indicator of therapeutic efficacy. A viral load > 102.5 copies/µg DNA be used as a diagnostic criterion for CAEBV infection^ref.
• NK cell-type CAEBV : HMB and high IgE

Symptoms & signs : liver dysfunction
Therapy : vidarabine
• T-cell type CAEBV : fever and high titers of EBV-related antibodies. Although not statistically significant, patients with T-cell type of CAEBV infection had higher IgG levels than those with NK-cell type.. It is possible that EBV-infected T cells become activated and release inflammatory cytokines such as IFN-g, IL-6, or TNF-a, resulting in severe inflammation and fever. In EBV-related hemophagocytic syndrome, it has been shown that viral-infected T cells release TNF-a and activate macrophages, resulting in massive hemophagocytosis^{ref1, ref2}. These activated T cells might induce polyclonal B-cell activation through cytokine release and thereby induce high levels of IgG and EBV-related antibodies. On the other hand, it is unclear why HMB and high IgE were observed in patients with NK-cell type of CAEBV infection. We should emphasize that determining the cell type is important in predicting disease prognosis, because the survival rates were different between the 2 groups. Differences in survival rates are particularly important in assessing treatment choices. T
It remains unclear whether these 2 manifestations of disease represent different entities or simply appear different because of the nature of the infected cells
• severe chronic active EBV-infection (SCAEBV) (late 1970s)

Symptoms & signs : similar to those of chronic fatigue syndrome (CFS) but more severe in degree (pancytopenia, high fever, massive splenomegaly) => poor prognosis
Therapy : effect of in vitro-generated autologous EBV-specific CTLs or LAK cells  is limited or deteriorative
Therapy : to date, a treatment for CAEBV infection has not yet been established. Antiviral or immunomodulating agents, such as acyclovir, gancyclovir, vidarabine, IFN-g, and IL-2, have been tried^{ref1, Wakiguchi H, 2022-2025, ref3, ref4}. Adoptive transfer of virus-specific CTLs to a patient with CAEBV infection has been reported^ref. However, most of these reports were anecdotal and there are few confirmatory reports. Immunochemotherapy consisting of etoposide, steroids, and cyclosporin A was proposed for use in patients with advanced CAEBV infection^ref, but no evidence of its efficacy is available. Recently, successful treatment of CAEBV infection by allogeneic bone marrow transplantation has been reported^ref. Viral loads decreased in all patients receiving transplants, some of whom appeared to be cured. However, HSCT constitutes a considerable risk, because 4 of 7 patients died after transplantation. Prospective studies analyzing a larger number of patients with CAEBV infection are necessary to confirm the efficacy of transplantation and to establish safer conditioning regimens.
• EBV-associated