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1.  Hydroa vacciniforme-like lymphoma of an adult: a case report with review of the literature 
Diagnostic Pathology  2013;8:72.
Virtual slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/7644172219178472
Hydroa vacciniforme-like lymphoma (HVL) is a rare type of Epstein-Barr virus (EBV)-positive lymphoma of cytotoxic T-cell or natural killer cell origin that mainly affect children, characterized by a vesicopapular skin eruption that clinically resemble hydroa vacciniforme (HV). In current study, we report an adult patient with the tumor. The patient presented similar morphologic, immunophenotypic and genotypic changes of the disease with that occurred in children, whereas clinically, he showed a prolonged clinical course without hepatosplenomegaly or generalized lymphadenopathy. Whether there are some differences in biologic behavior between children and adults still remains unknown and it is necessary to collect more data to observe and to investigate in the future.
doi:10.1186/1746-1596-8-72
PMCID: PMC3662161  PMID: 23635342
Hydroa vacciniforme-like lymphoma; Epstein-Barr virus; Adult
2.  Systemic lymphoma arising from hydroa vacciniforme-like lymphoma: report of two cases with review of literature 
Hydroa vacciniforme-like lymphoma (HVLL) is an extremely rare lymphoma described in children that occurs mainly in Asia and Latin American countries. It is an Epstein-Barr virus (EBV)-positive lymphoproliferative disease (LPD) characterized by a monoclonal proliferation of T or NK cells. In this study, we report the clinical and pathological features of two Chinese patients with HVLL showed T-cell phenotype expressing CD4. The two patients generally presented with similar clinical histories of waxing and waning ulcerative blistering lesions for ten years or more until progression to systemic lymphoma. One patient died two months after progression and another is alive with disease. In the two cases, persistence infection of EBV may be attributed to the disease progression, and systemic lymphoma arising from HVLL behaves in an aggressive fashion and is predisposing to chemotherapeutic agent resistance.
PMCID: PMC4203271  PMID: 25337300
Hydroa vacciniforme; lymphoma; Epstein-Barr virus infections
3.  EBV Chronic Infections 
The infection from Epstein-Barr virus (EBV) or virus of infectious mononucleosis, together with other herpes viruses’ infections, represents a prototype of persistent viral infections characterized by the property of the latency. Although the reactivations of the latent infection are associated with the resumption of the viral replication and eventually with the “shedding”, it is still not clear if this virus can determine chronic infectious diseases, more or less evolutive. These diseases could include some pathological conditions actually defined as “idiopathic”and characterized by the “viral persistence” as the more credible pathogenetic factor. Among the so-called idiopathic syndromes, the “chronic fatigue syndrome” (CFS) aroused a great interest around the eighties of the last century when, just for its relationship with EBV, it was called “chronic mononucleosis” or “chronic EBV infection”.
Today CFS, as defined in 1994 by the CDC of Atlanta (USA), really represents a multifactorial syndrome characterized by a chronic course, where reactivation and remission phases alternate, and by a good prognosis. The etiopathogenetic role of EBV is demonstrated only in a well-examined subgroup of patients, while in most of the remaining cases this role should be played by other infectious agents - able to remain in a latent or persistent way in the host – or even by not infectious agents (toxic, neuroendocrine, methabolic, etc.). However, the pathogenetic substrate of the different etiologic forms seems to be the same, much probably represented by the oxidative damage due to the release of pro-inflammatory cytokines as a response to the triggering event (infectious or not infectious).
Anyway, recently the scientists turned their’s attention to the genetic predisposition of the subjects affected by the syndrome, so that in the last years the genetic studies, together with those of molecular biology, received a great impulse. Thanks to both these studies it was possibile to confirm the etiologic links between the syndrome and EBV or other herpesviruses or other persistent infectious agents.
The mechanisms of EBV latency have been carefully examined both because they represent the virus strategy to elude the response of the immune system of the host, and because they are correlated with those oncologic conditions associated to the viral persistence, particularly lymphomas and lymphoproliferative disorders. Just these malignancies, for which a pathogenetic role of EBV is clearly documented, should represent the main clinical expression of a first group of chronic EBV infections characterized by a natural history where the neoplastic event aroused from the viral persistence in the resting B cells for all the life, from the genetic predisposition of the host and from the oncogenic potentialities of the virus that chronically persists and incurs reactivations.
Really, these oncological diseases should be considered more complications than chronic forms of the illness, as well as other malignancies for which a viral – or even infectious - etiology is well recognized. The chronic diseases, in fact, should be linked in a pathogenetic and temporal way to the acute infection, from whom start the natural history of the following disease. So, as for the chronic liver diseases from HBV and HCV, it was conied the acronym of CAEBV (Chronic Active EBV infection), distinguishing within these pathologies the more severe forms (SCAEBV) mostly reported in Far East and among children or adolescents. Probably only these forms have to be considered expressions of a chronic EBV infection “sensu scrictu”, together with those forms of CFS where the etiopathogenetic and temporal link with the acute EBV infection is well documented. As for CFS, also for CAEBV the criteria for a case definition were defined, even on the basis of serological and virological findings. However, the lymphoproliferative disorders are excluded from these forms and mantain their nosographic (e.g. T or B cell or NK type lymphomas) and pathogenetic collocation, even when they occur within chronic forms of EBV infection. In the pathogenesis, near to the programs of latency of the virus, the genetic and environmental factors, independent from the real natural history of EBV infection, play a crucial role.
Finally, it was realized a review of cases - not much numerous in literature – of chronic EBV infection associated to chronic liver and neurological diseases, where the modern techniques of molecular biology should be useful to obtain a more exact etiologic definition, not always possibile to reach in the past.
The wide variety of clinical forms associated to the EBV chronic infection makes difficult the finding of a univocal pathogenetic link. There is no doubt, however, that a careful examination of the different clinical forms described in this review should be useful to open new horizons to the study of the persistent viral infections and the still not well cleared pathologies that they can induce in the human host.
doi:10.4084/MJHID.2010.022
PMCID: PMC3033110  PMID: 21415952
4.  A Case of Epstein-Barr Virus-associated Hydroa Vacciniforme 
Annals of Dermatology  2009;21(2):209-212.
Hydroa vacciniforme (HV) is a photosensitivity disorder characterized by recurrent necrotic vesiculopapules on sun-exposed areas, which heal spontaneously during adolescence. Recently, an association has been reported between latent Epstein-Barr virus (EBV) infection and atypical HV-like eruption and malignant potential. However, latent EBV infection has also been reported in the setting of typical HV. An 11-year-old girl presented with recurrent, scattered, discrete vesicular eruptions with scarring on the face and the extensor surfaces of both forearms. In-situ hybridization was carried out to detect latent EBV infection. Based on the clinical and histopathological findings, typical EBV-associated HV was suspected.
doi:10.5021/ad.2009.21.2.209
PMCID: PMC2861220  PMID: 20523790
Epstein-Barr virus; Hydroa vacciniforme
5.  HIV Patients Developing Primary CNS Lymphoma Lack EBV-Specific CD4+ T Cell Function Irrespective of Absolute CD4+ T Cell Counts 
PLoS Medicine  2007;4(3):e96.
Background
In chronic HIV infection, antiretroviral therapy–induced normalization of CD4+ T cell counts (immune reconstitution [IR]) is associated with a decreased incidence of opportunistic diseases. However, some individuals remain at risk for opportunistic diseases despite prolonged normalization of CD4+ T cell counts. Deficient Epstein-Barr virus (EBV)-specific CD4+ T cell function may explain the occurrence of EBV-associated opportunistic malignancy—such as primary central nervous system (PCNS) lymphoma—despite recovery of absolute CD4+ T cell counts.
Methods and Findings
Absolute CD4+ T cell counts and EBV-specific CD4+ T cell-dependent interferon-γ production were assessed in six HIV-positive individuals prior to development of PCNS lymphoma (“cases”), and these values were compared with those in 16 HIV-infected matched participants with no sign of EBV-associated pathology (“matched controls”) and 11 nonmatched HIV-negative blood donors. Half of the PCNS lymphoma patients fulfilled IR criteria (defined here as CD4+ T cell counts ≥500/μl blood). EBV-specific CD4+ T cells were assessed 0.5–4.7 y prior to diagnosis of lymphoma. In 0/6 cases versus 13/16 matched controls an EBV-specific CD4+ T cell response was detected (p = 0.007; confidence interval for odds ratio [0–0.40]). PCNS lymphoma patients also differed with regards to this response significantly from HIV-negative blood donors (p < 0.001, confidence interval for odds ratio [0–0.14]), but there was no evidence for a difference between HIV-negative participants and the HIV-positive matched controls (p = 0.47).
Conclusions
Irrespective of absolute CD4+ T cell counts, HIV-positive patients who subsequently developed PCNS lymphoma lacked EBV-specific CD4+ T cell function. Larger, ideally prospective studies are needed to confirm these preliminary data, and clarify the impact of pathogen-specific versus surrogate marker-based assessment of IR on clinical outcome.
In a case-control study from the Swiss HIV cohort, Hess and colleagues report that T-helper responses against Epstein-Barr virus are specifically absent in patients developing CNS lymphoma.
Editors' Summary
Background.
AIDS causes disease by inactivating the body's immune responses. Most severely affected are the white blood cells known as T lymphocytes, particularly the CD4+ T cells that recognize infection and enable other cells of the immune system to respond. Advanced HIV infection, marked by very low numbers of CD4+ cells, is associated with a variety of infections and tumors that are rarely seen in people with intact immune systems. People with advanced HIV who receive highly active antiretroviral treatment (HAART) tend to have increases in their CD4+ cell counts and lose their susceptibility to these so-called opportunistic infections and cancers. For several common opportunistic infections, it is considered safe to discontinue preventive antibiotics after a patient's total CD4+ cell count has returned to normal levels on HAART. Some treated individuals, however, will develop these conditions even after their CD4+ cell counts have returned to normal levels. The reason this happens is unclear.
Why Was This Study Done?
For several years, scientists have speculated that susceptibility to a given opportunistic infection might be due not simply to low total CD4+ cells, but to loss of the specific CD4+ cells that recognize the infection in question. If this theory is correct, then those individuals who develop an opportunistic condition after their total CD4+ cell counts return to normal might be missing the specific cells that respond to the microbe causing the condition. The researchers wanted to test this theory in HIV patients with a brain tumor called primary central nervous system lymphoma (PCNS lymphoma). The Epstein-Barr virus (EBV), which causes mononucleosis in the general population, has been shown to be a cause of PCNS lymphoma in people with AIDS.
What Did the Researchers Do and Find?
The researchers studied patients who developed PCNS lymphoma while enrolled in the Swiss HIV Cohort, an ongoing study that has enrolled more than 14,000 people. A large cohort was needed to address this question because PCNS lymphoma is uncommon, and indeed only six patients with a confirmed diagnosis were identified. Because they had been followed as part of the cohort study, these patients had given blood samples that could be tested in retrospect. Three of these patients had low CD4+ cell counts prior to lymphoma diagnosis and three had normal CD4+ cell counts, but CD4 responses specifically against EBV were absent or very low in all six patients before they were diagnosed with PCNS lymphoma. The researchers also studied a comparison group of cohort participants with comparable CD4+ cell counts but no PCNS lymphoma, and found that 13/16 of those participants did have CD4 responses to EBV.
What Do These Findings Mean?
These results support the idea that the action of EBV-specific CD4+ cells, rather than a given level of total CD4+ cells, is needed to prevent PCNS lymphoma. Because only a small number of cases were identified, this must be considered a preliminary result. Given the rarity of PCNS lymphoma, however, especially in people receiving HAART, it seems unlikely that a larger cohort will be available in the near future to provide a more definitive conclusion. Based on this result, it may be useful to perform similar studies of other opportunistic infections. If a “gap” in the CD4+ cell response can be shown to increase the risk of a specific condition, it may become appropriate to test specific CD4 responses before deciding to discontinue preventive treatment as CD4+ cell counts increase on HAART.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040096.
Read the accompanying Perspective by Mark Jacobson, MD
The Swiss Cohort Study Web site contains information on related research projects
The UCSF Center for HIV Information's HIV InSite includes resources on HIV immunology and opportunistic infections
doi:10.1371/journal.pmed.0040096
PMCID: PMC1831733  PMID: 17388662
6.  Adoptive Transfer of EBV Specific CD8+ T Cell Clones Can Transiently Control EBV Infection in Humanized Mice 
PLoS Pathogens  2014;10(8):e1004333.
Epstein Barr virus (EBV) infection expands CD8+ T cells specific for lytic antigens to high frequencies during symptomatic primary infection, and maintains these at significant numbers during persistence. Despite this, the protective function of these lytic EBV antigen-specific cytotoxic CD8+ T cells remains unclear. Here we demonstrate that lytic EBV replication does not significantly contribute to virus-induced B cell proliferation in vitro and in vivo in a mouse model with reconstituted human immune system components (huNSG mice). However, we report a trend to reduction of EBV-induced lymphoproliferation outside of lymphoid organs upon diminished lytic replication. Moreover, we could demonstrate that CD8+ T cells against the lytic EBV antigen BMLF1 can eliminate lytically replicating EBV-transformed B cells from lymphoblastoid cell lines (LCLs) and in vivo, thereby transiently controlling high viremia after adoptive transfer into EBV infected huNSG mice. These findings suggest a protective function for lytic EBV antigen-specific CD8+ T cells against EBV infection and against virus-associated tumors in extra-lymphoid organs. These specificities should be explored for EBV-specific vaccine development.
Author Summary
Epstein Barr virus persistently infects more than 90% of the human adult population. While fortunately carried as an asymptomatic chronic infection in most individuals, it causes B cell lymphomas and carcinomas in some patients. Symptomatic primary EBV infection, called infectious mononucleosis, predisposes for some of these malignancies and is characterized by massive expansions of cytotoxic T cells, which are mostly directed against lytic EBV antigens that are expressed during virus particle production. Therefore, we investigated the protective role of lytic EBV antigen specific T cells during EBV infection and the contribution of lytic EBV infection to virus-associated tumor formation. We found that lytic EBV antigen specific T cells kill B cells with lytic virus replication and might thereby transiently control EBV infection in mice with human immune system components. Furthermore, we observed that EBV associated B cell tumors outside secondary lymphoid organs may require lytic replication for efficient formation. Thus, we suggest that lytic EBV antigens should be explored for vaccination against symptomatic EBV infection and EBV associated extra-lymphoid tumors.
doi:10.1371/journal.ppat.1004333
PMCID: PMC4148450  PMID: 25165855
7.  An Epstein-Barr Virus Encoded Inhibitor of Colony Stimulating Factor-1 Signaling Is an Important Determinant for Acute and Persistent EBV Infection 
PLoS Pathogens  2012;8(12):e1003095.
Acute Epstein-Barr virus (EBV) infection is the most common cause of Infectious Mononucleosis. Nearly all adult humans harbor life-long, persistent EBV infection which can lead to development of cancers including Hodgkin Lymphoma, Burkitt Lymphoma, nasopharyngeal carcinoma, gastric carcinoma, and lymphomas in immunosuppressed patients. BARF1 is an EBV replication-associated, secreted protein that blocks Colony Stimulating Factor 1 (CSF-1) signaling, an innate immunity pathway not targeted by any other virus species. To evaluate effects of BARF1 in acute and persistent infection, we mutated the BARF1 homologue in the EBV-related herpesvirus, or lymphocryptovirus (LCV), naturally infecting rhesus macaques to create a recombinant rhLCV incapable of blocking CSF-1 (ΔrhBARF1). Rhesus macaques orally challenged with ΔrhBARF1 had decreased viral load indicating that CSF-1 is important for acute virus infection. Surprisingly, ΔrhBARF1 was also associated with dramatically lower virus setpoints during persistent infection. Normal acute viral load and normal viral setpoints during persistent rhLCV infection could be restored by Simian/Human Immunodeficiency Virus-induced immunosuppression prior to oral inoculation with ΔrhBARF1 or infection of immunocompetent animals with a recombinant rhLCV where the rhBARF1 was repaired. These results indicate that BARF1 blockade of CSF-1 signaling is an important immune evasion strategy for efficient acute EBV infection and a significant determinant for virus setpoint during persistent EBV infection.
Author Summary
Epstein-Barr virus (EBV) is a herpesvirus that persistently infects nearly all humans by adulthood. Acute and persistent phases of EBV infection are associated with a variety of human diseases, including infectious mononucleosis and cancer. To investigate how EBV interacts with the host to successfully establish acute and persistent infection, we combined the power of the rhesus macaque animal model for EBV infection with genetic engineering of the EBV-related herpesvirus, or lymphocryptovirus (LCV), that naturally infects rhesus macaques. We created a recombinant rhLCV carrying a mutated EBV BARF1 homologue, a replication-associated viral protein that is secreted and blocks Colony Stimulating Factor-1 (CSF-1) signaling, a cytokine important for innate immunity. Oral inoculation of rhesus macaques showed that the virus' ability to block CSF-1 was important for achieving the normally high viral loads during acute infection, and surprisingly, was also needed to establish normal levels of virus infection, or viral setpoint, during persistent infection. These studies show that virus-mediated interruption of innate immunity is critical for both acute and persistent phases of EBV infection. Understanding how EBV successfully infects humans and how the natural history of EBV infection can be disrupted will aid in development of vaccines to prevent EBV-associated diseases.
doi:10.1371/journal.ppat.1003095
PMCID: PMC3531511  PMID: 23300447
8.  Epstein-Barr virus-driven lymphomagenesis in the context of human immunodeficiency virus type 1 infection 
Epstein–Barr virus (EBV) is a ubiquitous human γ-herpes virus which establishes a life-long asymptomatic infection in immunocompetent hosts. In human immunodeficiency virus type 1 (HIV-1) infected patients, the impaired immunosurveillance against EBV may favor the development of EBV-related diseases, ranging from lymphoproliferative disorders to B cell non-Hodgkin’s lymphomas (NHL). Antiretroviral therapy (ART) has significantly modified the natural course of HIV-1 infection, resulting in decreased HIV-1 plasmaviremia, increased CD4 lymphocytes, and decreased opportunistic infections, indicating a restoration of immune functions. However, the impact of ART appears to be less favorable on EBV-related malignancies than on other AIDS-defining tumors, such as Kaposi’s sarcoma, and NHL remains the most common cancer during the ART era. EBV-driven tumors are associated with selective expression of latent oncogenic proteins, but uncontrolled lytic cycle with virus replication and/or reactivation may favor cell transformation, at least in the early phases. Several host’s factors may promote EBV reactivation and replication; besides immunodepression, inflammation/chronic immune stimulation may play an important role. Microbial pathogen-associated molecular patterns and endogenous damage-associated molecular patterns, through Toll-like receptors, activate the immune system and may promote EBV reactivation and/or polyclonal expansion of EBV-infected cells. A body of evidence suggests that chronic immune stimulation is a hallmark of HIV-1 pathogenesis and may persist even in ART-treated patients. This review focuses on lymphomagenesis driven by EBV both in the context of the natural history of HIV-1 infection and in ART-treated patients. Understanding the mechanisms involved in the expansion of EBV-infected cells is a premise for the identification of prognostic markers of EBV-associated malignancies.
doi:10.3389/fmicb.2013.00311
PMCID: PMC3799006  PMID: 24151490
EBV; HIV-1; B cell activation; chronic immune activation; EBV-related malignancies; antiretroviral therapy; EBV lytic reactivation
9.  The Spectrum of Epstein-Barr Virus-Associated Lymphoproliferative Disease in Korea: Incidence of Disease Entities by Age Groups 
Journal of Korean Medical Science  2008;23(2):185-192.
This study is to identify the spectrum of Epstein-Barr virus (EBV)-positive lymphoproliferative diseases (LPD) and relationships between these diseases in Korea. The EBV status and clinicopathology of 764 patients, including acute EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), chronic active EBV (CAEBV) infections, B-LPD arising in chronic latent EBV infection, T & natural killer (NK) cell non-Hodgkin's lymphomas (NHL), B-NHLs, and Hodgkin's lymphomas (HD), were analyzed. T or NK cell NHLs were the most common forms of EBV-positive NHLs (107/167, 64%); among these, nasal-type NK/T cell lymphomas were the most common (89/107, 83%). According to the age, Burkitt's lymphoma was the most common in early childhood; in teenagers, chronic (active) EBV infection-associated LPD was the most common type. The incidence of NK/T cell lymphoma began to increase from the twenties and formed the major type of EBV-associated tumor throughout life. Diffuse large B cell lymphoma formed the major type in the sixties and seventies. In conclusion, primary infections in early childhood are complicated by the development of CAEBV infections that are main predisposing factors for EBV-associated T or NK cell malignancies in young adults. In old patients, decreased immunity associated with old age and environmental cofactors may provoke the development of peripheral T cell lymphoma, unspecified, and diffuse large B cell lymphoma.
doi:10.3346/jkms.2008.23.2.185
PMCID: PMC2526432  PMID: 18436998
Lymphoma; Epstein-Barr Virus; Lymphohistiocytosis, Hemophagocytic
10.  Novel Mouse Xenograft Models Reveal a Critical Role of CD4+ T Cells in the Proliferation of EBV-Infected T and NK Cells 
PLoS Pathogens  2011;7(10):e1002326.
Epstein-Barr virus (EBV), a ubiquitous B-lymphotropic herpesvirus, ectopically infects T or NK cells to cause severe diseases of unknown pathogenesis, including chronic active EBV infection (CAEBV) and EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). We developed xenograft models of CAEBV and EBV-HLH by transplanting patients' PBMC to immunodeficient mice of the NOD/Shi-scid/IL-2Rγnull strain. In these models, EBV-infected T, NK, or B cells proliferated systemically and reproduced histological characteristics of the two diseases. Analysis of the TCR repertoire expression revealed that identical predominant EBV-infected T-cell clones proliferated in patients and corresponding mice transplanted with their PBMC. Expression of the EBV nuclear antigen 1 (EBNA1), the latent membrane protein 1 (LMP1), and LMP2, but not EBNA2, in the engrafted cells is consistent with the latency II program of EBV gene expression known in CAEBV. High levels of human cytokines, including IL-8, IFN-γ, and RANTES, were detected in the peripheral blood of the model mice, mirroring hypercytokinemia characteristic to both CAEBV and EBV-HLH. Transplantation of individual immunophenotypic subsets isolated from patients' PBMC as well as that of various combinations of these subsets revealed a critical role of CD4+ T cells in the engraftment of EBV-infected T and NK cells. In accordance with this finding, in vivo depletion of CD4+ T cells by the administration of the OKT4 antibody following transplantation of PBMC prevented the engraftment of EBV-infected T and NK cells. This is the first report of animal models of CAEBV and EBV-HLH that are expected to be useful tools in the development of novel therapeutic strategies for the treatment of the diseases.
Author Summary
Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that infects more than 90% of the adult human population in the world. EBV usually infects B lymphocytes and does not produce symptoms in infected individuals, but in rare occasions it infects T or NK lymphocytes and causes severe diseases such as chronic active EBV infection (CAEBV) and EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). We developed mouse models of these two human diseases in which EBV-infected T or NK lymphocytes proliferate in mouse tissues and reproduce human pathologic conditions such as overproduction of small proteins called “cytokines” that produce inflammatory responses in the body. These mouse models are thought to be very useful for the elucidation of the pathogenesis of CAEBV and EBV-HLH as well as for the development of therapeutic strategies for the treatment of these diseases. Experiments with the models demonstrated that a subset of lymphocytes called CD4-positive lymphocytes are essential for the proliferation of EBV-infected T and NK cells. This result implies that removal of CD4-positive lymphocytes or suppression of their functions may be an effective strategy for the treatment of CAEBV and EBV-HLH.
doi:10.1371/journal.ppat.1002326
PMCID: PMC3197618  PMID: 22028658
11.  Exhausted Cytotoxic Control of Epstein-Barr Virus in Human Lupus 
PLoS Pathogens  2011;7(10):e1002328.
Systemic Lupus Erythematosus (SLE) pathology has long been associated with an increased Epstein-Barr Virus (EBV) seropositivity, viremia and cross-reactive serum antibodies specific for both virus and self. It has therefore been postulated that EBV triggers SLE immunopathology, although the mechanism remains elusive. Here, we investigate whether frequent peaks of EBV viral load in SLE patients are a consequence of dysfunctional anti-EBV CD8+ T cell responses. Both inactive and active SLE patients (n = 76 and 42, respectively), have significantly elevated EBV viral loads (P = 0.003 and 0.002, respectively) compared to age- and sex-matched healthy controls (n = 29). Interestingly, less EBV-specific CD8+ T cells are able to secrete multiple cytokines (IFN-γ, TNF-α, IL-2 and MIP-1β) in inactive and active SLE patients compared to controls (P = 0.0003 and 0.0084, respectively). Moreover, EBV-specific CD8+ T cells are also less cytotoxic in SLE patients than in controls (CD107a expression: P = 0.0009, Granzyme B release: P = 0.0001). Importantly, cytomegalovirus (CMV)-specific responses were not found significantly altered in SLE patients. Furthermore, we demonstrate that EBV-specific CD8+ T cell impairment is a consequence of their Programmed Death 1 (PD-1) receptor up-regulation, as blocking this pathway reverses the dysfunctional phenotype. Finally, prospective monitoring of lupus patients revealed that disease flares precede EBV reactivation. In conclusion, EBV-specific CD8+ T cell responses in SLE patients are functionally impaired, but EBV reactivation appears to be an aggravating consequence rather than a cause of SLE immunopathology. We therefore propose that autoimmune B cell activation during flares drives frequent EBV reactivation, which contributes in a vicious circle to the perpetuation of immune activation in SLE patients.
Author Summary
Systemic Lupus Erythematosus (SLE) has been associated with Epstein-Barr Virus (EBV) infection for decades, however the mechanistic links have remained elusive. Most human adults are infected by EBV and carry the virus for life without clinical symptoms. However, for unknown reasons EBV induces infectious mononucleosis in some individuals, during which cross-reactive antibodies specific for both virus and self have been detected. Interestingly, such cross-reactive antibodies are also frequently found in SLE patients. Since, EBV seropositivity and viremia are more frequent in SLE patients than in healthy individuals, it has been postulated that EBV trigger autoimmunity. Here we show that SLE patients are indeed less capable of controlling EBV viremia, since their EBV-specific CD8+ T cells have diminished capacity to secrete effector molecules (e.g. cytokines and chemokines) and to kill EBV-infected targets as a consequence of their Programmed Death 1 (PD-1) receptor up-regulation. Longitudinal studies further reveal that disease flares precede EBV viremia. Thus, contrary to expectations, EBV reactivation appears to be an aggravating consequence, rather than a cause, of SLE immunopathology. Our results pave the way for immunological interventions that restore the host-EBV balance, which may result in decreased levels of aggravating cross-reactive antibodies and ultimately be beneficial to SLE patients.
doi:10.1371/journal.ppat.1002328
PMCID: PMC3197610  PMID: 22028659
12.  Hydroa Vacciniforme-Like Cutaneous T-cell Lymphoma 
Indian Journal of Dermatology  2014;59(1):91-93.
A 14-year-old Chinese girl had a 6-year history of recurrent lesions on her head, face, and limbs. Epstein-Barr virus (EBV)-IgM was positive. Histopathological findings revealed focal lymphocyte invasion in subcutaneous panniculus adiposus, mainly surrounding the blood vessels. Immunohistochemistry showed CD3+, CD4+, CD5+, CD8+, TIA-1+, GrB+, CD56-, and L26-. In situ hybridization staining for EBV-encoded small nonpolyadenylated RNA (EBER)-1 was positive. The patient showed significant improvement in clinical symptoms after being treated with acyclovir and IFN-α in this patient.
doi:10.4103/0019-5154.123516
PMCID: PMC3884939  PMID: 24470671
Epstein-Barr virus; hydroa vacciniforme-like; lymlphoma; T-cell
13.  Epstein–Barr virus-associated lymphoproliferative disease in non-immunocompromised hosts: a status report and summary of an international meeting, 8–9 September 2008 
Annals of Oncology  2009;20(9):1472-1482.
Background: Recently novel Epstein–Barr virus (EBV) lymphoproliferative diseases (LPDs) have been identified in non-immunocompromised hosts, both in Asia and Western countries. These include aggressive T-cell and NK-cell LPDs often subsumed under the heading of chronic active Epstein–Barr virus (CAEBV) infection and EBV-driven B-cell LPDs mainly affecting the elderly.
Design: To better define the pathogenesis, classification, and treatment of these disorders, participants from Asia, The Americas, Europe, and Australia presented clinical and experimental data at an international meeting.
Results: The term systemic EBV-positive T-cell LPD, as adopted by the WHO classification, is preferred as a pathological classification over CAEBV (the favored clinical term) for those cases that are clonal. The disease has an aggressive clinical course, but may arise in the background of CAEBV. Hydroa vacciniforme (HV) and HV-like lymphoma represent a spectrum of clonal EBV-positive T-cell LPDs, which have a more protracted clinical course; spontaneous regression may occur in adult life. Severe mosquito bite allergy is a related syndrome usually of NK cell origin. Immune senescence in the elderly is associated with both reactive and neoplastic EBV-driven LPDs, including EBV-positive diffuse large B-cell lymphomas.
Conclusion: The participants proposed an international consortium to facilitate further clinical and biological studies of novel EBV-driven LPDs.
doi:10.1093/annonc/mdp064
PMCID: PMC2731018  PMID: 19515747
chronic active EBV infection; diffuse large B-cell lymphoma; hemophagocytic syndrome; hydroa vacciniforme; immune senescence; senile EBV-positive lymphoproliferative disease; systemic EBV-positive lymphoproliferative disease
14.  A Genome-Wide Integrative Genomic Study Localizes Genetic Factors Influencing Antibodies against Epstein-Barr Virus Nuclear Antigen 1 (EBNA-1) 
PLoS Genetics  2013;9(1):e1003147.
Infection with Epstein-Barr virus (EBV) is highly prevalent worldwide, and it has been associated with infectious mononucleosis and severe diseases including Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal lymphoma, and lymphoproliferative disorders. Although EBV has been the focus of extensive research, much still remains unknown concerning what makes some individuals more sensitive to infection and to adverse outcomes as a result of infection. Here we use an integrative genomics approach in order to localize genetic factors influencing levels of Epstein Barr virus (EBV) nuclear antigen-1 (EBNA-1) IgG antibodies, as a measure of history of infection with this pathogen, in large Mexican American families. Genome-wide evidence of both significant linkage and association was obtained on chromosome 6 in the human leukocyte antigen (HLA) region and replicated in an independent Mexican American sample of large families (minimum p-value in combined analysis of both datasets is 1.4×10−15 for SNPs rs477515 and rs2516049). Conditional association analyses indicate the presence of at least two separate loci within MHC class II, and along with lymphocyte expression data suggest genes HLA-DRB1 and HLA-DQB1 as the best candidates. The association signals are specific to EBV and are not found with IgG antibodies to 12 other pathogens examined, and therefore do not simply reveal a general HLA effect. We investigated whether SNPs significantly associated with diseases in which EBV is known or suspected to play a role (namely nasopharyngeal lymphoma, Hodgkin lymphoma, systemic lupus erythematosus, and multiple sclerosis) also show evidence of associated with EBNA-1 antibody levels, finding an overlap only for the HLA locus, but none elsewhere in the genome. The significance of this work is that a major locus related to EBV infection has been identified, which may ultimately reveal the underlying mechanisms by which the immune system regulates infection with this pathogen.
Author Summary
Many factors influence individual differences in susceptibility to infectious disease, including genetic factors of the host. Here we use several genome-wide investigative tools (linkage, association, joint linkage and association, and the analysis of gene expression data) to search for host genetic factors influencing Epstein-Barr virus (EBV) infection. EBV is a human herpes virus that infects up to 90% of adults worldwide, infection with which has been associated with severe complications including malignancies and autoimmune disorders. In a sample of >1,300 Mexican American family members, we found significant evidence of association of anti–EBV antibody levels with loci on chromosome 6 in the human leukocyte antigen region, which contains genes related to immune function. The top two independent loci in this region were HLA-DRB1 and HLA-DQB1, both of which are involved in the presentation of foreign antigens to T cells. This finding was specific to EBV and not to 12 other pathogens we examined. We also report an overlap of genetic factors influencing both EBV antibody level and EBV–related cancers and autoimmune disorders. This work demonstrates the presence of EBV susceptibility loci and provides impetus for further investigation to better understand the underlying mechanisms related to differences in disease progression among individuals infected with this pathogen.
doi:10.1371/journal.pgen.1003147
PMCID: PMC3542101  PMID: 23326239
15.  A Viral microRNA Cluster Strongly Potentiates the Transforming Properties of a Human Herpesvirus 
PLoS Pathogens  2011;7(2):e1001294.
Epstein-Barr virus (EBV), an oncogenic human herpesvirus, induces cell proliferation after infection of resting B lymphocytes, its reservoir in vivo. The viral latent proteins are necessary for permanent B cell growth, but it is unknown whether they are sufficient. EBV was recently found to encode microRNAs (miRNAs) that are expressed in infected B cells and in some EBV-associated lymphomas. EBV miRNAs are grouped into two clusters located either adjacent to the BHRF1 gene or in introns contained within the viral BART transcripts. To understand the role of the BHRF1 miRNA cluster, we have constructed a virus mutant that lacks all its three members (Δ123) and a revertant virus. Here we show that the B cell transforming capacity of the Δ123 EBV mutant is reduced by more than 20-fold, relative to wild type or revertant viruses. B cells exposed to the knock-out virus displayed slower growth, and exhibited a two-fold reduction in the percentage of cells entering the cell cycle S phase. Furthermore, they displayed higher latent gene expression levels and latent protein production than their wild type counterparts. Therefore, the BHRF1 miRNAs accelerate B cell expansion at lower latent gene expression levels. Thus, this miRNA cluster simultaneously enhances expansion of the virus reservoir and reduces the viral antigenic load, two features that have the potential to facilitate persistence of the virus in the infected host. Thus, the EBV BHRF1 miRNAs may represent new therapeutic targets for the treatment of some EBV-associated lymphomas.
Author Summary
To persist in their hosts, herpes viruses must avoid recognition by the host's immune system. Down-regulation of viral antigen production through expression of viral miRNAs is a particularly elegant mechanism as these genetic elements do not encode proteins and remain therefore invisible to the immune system. Upon primary infection, Epstein-Barr virus (EBV) colonizes B cells and, through expression of its latent proteins, induces their continuous proliferation. The resulting expansion of infected B cells elicits a T cell response directed against the latent proteins that results in their elimination. Therefore, rapid proliferation of infected B cells, combined with reduced latent protein production, would facilitate establishment of EBV's viral reservoir before mounting of the immune response. Here, we find that a cluster of three microRNAs encoded near the EBV BHRF1 gene is crucial for efficient B cell transformation. In the absence of these genetic elements, infected B cells grow markedly more slowly. Furthermore, B cells exposed to an EBV mutant that lacks the BHRF1 microRNA cluster produced more latent proteins. Thus, the BHRF1 microRNA cluster possesses properties that potentiate EBV's oncogenic properties and therefore facilitate expansion of the EBV B cell reservoir.
doi:10.1371/journal.ppat.1001294
PMCID: PMC3040666  PMID: 21379335
16.  Soluble Rhesus Lymphocryptovirus gp350 Protects against Infection and Reduces Viral Loads in Animals that Become Infected with Virus after Challenge 
PLoS Pathogens  2011;7(10):e1002308.
Epstein-Barr virus (EBV) is a human lymphocryptovirus that is associated with several malignancies. Elevated EBV DNA in the blood is observed in transplant recipients prior to, and at the time of post-transplant lymphoproliferative disease; thus, a vaccine that either prevents EBV infection or lowers the viral load might reduce certain EBV malignancies. Two major approaches have been suggested for an EBV vaccine- immunization with either EBV glycoprotein 350 (gp350) or EBV latency proteins (e.g. EBV nuclear antigens [EBNAs]). No comparative trials, however, have been performed. Rhesus lymphocryptovirus (LCV) encodes a homolog for each gene in EBV and infection of monkeys reproduces the clinical, immunologic, and virologic features of both acute and latent EBV infection. We vaccinated rhesus monkeys at 0, 4 and 12 weeks with (a) soluble rhesus LCV gp350, (b) virus-like replicon particles (VRPs) expressing rhesus LCV gp350, (c) VRPs expressing rhesus LCV gp350, EBNA-3A, and EBNA-3B, or (d) PBS. Animals vaccinated with soluble gp350 produced higher levels of antibody to the glycoprotein than those vaccinated with VRPs expressing gp350. Animals vaccinated with VRPs expressing EBNA-3A and EBNA-3B developed LCV-specific CD4 and CD8 T cell immunity to these proteins, while VRPs expressing gp350 did not induce detectable T cell immunity to gp350. After challenge with rhesus LCV, animals vaccinated with soluble rhesus LCV gp350 had the best level of protection against infection based on seroconversion, viral DNA, and viral RNA in the blood after challenge. Surprisingly, animals vaccinated with gp350 that became infected had the lowest LCV DNA loads in the blood at 23 months after challenge. These studies indicate that gp350 is critical for both protection against infection with rhesus LCV and for reducing the viral load in animals that become infected after challenge. Our results suggest that additional trials with soluble EBV gp350 alone, or in combination with other EBV proteins, should be considered to reduce EBV infection or virus-associated malignancies in humans.
Author Summary
Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and is associated with several cancers. Presently there is no licensed vaccine to prevent EBV diseases. Two types of candidate vaccines are under development; one involves immunization with the major glycoprotein (gp350) on the outside of the virus, while the other involves vaccination with EBV proteins expressed during latency. We compared these two types of candidate vaccines in a rhesus monkey model of EBV and found that the gp350 vaccine induced better protection from infection. In addition, animals that received the rhesus EBV glycoprotein and became infected had a lower level of rhesus EBV DNA in the blood at 23 months after challenge than animals that received the rhesus EBV latency protein vaccine that subsequently were infected. Since levels of EBV DNA in the blood have been predictive for EBV lymphomas in transplant patients, the ability of rhesus EBV gp350 to reduce levels of rhesus EBV in the blood after infection suggests the EBV gp350 could have a role in reducing certain EBV-associated cancers. This is the first test of candidate vaccines in the rhesus monkey model of EBV and shows that this model should be useful in further evaluation of EBV vaccines.
doi:10.1371/journal.ppat.1002308
PMCID: PMC3197588  PMID: 22028652
17.  Chronic Herpesvirus Reactivation Occurs in Aging 
Experimental gerontology  2007;42(6):563-570.
The aged immune system is characterized by clonal expansions of CD8+ T cells of which a substantial portion are directed against Epstein-Barr virus (EBV) and cytomegalovirus (CMV). It is unknown if these expansions represent increased viral reactivation or simply reflect an accumulation over time. We investigated herpesvirus reactivation in young and old subjects co-infected with CMV and EBV. Using molecular and serological techniques, we found significant increases in both the frequency and magnitude of EBV and CMV reactivation in elderly subjects. CMV DNA was frequently detected in the urine of elderly subjects; EBV load in peripheral blood was also significantly increased. Notably, EBV DNA in plasma was detected in a majority of the elderly subjects which was supported by frequent transcription of late structural genes. Furthermore, CD8+ T cells specific for EBV structural antigens were detected in samples from the elderly. Samples from our younger control group were negative for EBV DNA in plasma, CMV DNA in urine, expression of structural transcripts, and lacked CD8+ T cells specific for EBV structural antigens. These findings indicate that the aged immune system is no longer able to control EBV and CMV reactivation that could now be characterized as chronic instead of latent.
doi:10.1016/j.exger.2007.01.005
PMCID: PMC1992441  PMID: 17337145
aging; herpesvirus; Epstein-Barr Virus; Cytomegalovirus
18.  Tonsilar NK Cells Restrict B Cell Transformation by the Epstein-Barr Virus via IFN-γ 
PLoS Pathogens  2008;4(2):e27.
Cells of the innate immune system act in synergy to provide a first line of defense against pathogens. Here we describe that dendritic cells (DCs), matured with viral products or mimics thereof, including Epstein-Barr virus (EBV), activated natural killer (NK) cells more efficiently than other mature DC preparations. CD56brightCD16− NK cells, which are enriched in human secondary lymphoid tissues, responded primarily to this DC activation. DCs elicited 50-fold stronger interferon-γ (IFN-γ) secretion from tonsilar NK cells than from peripheral blood NK cells, reaching levels that inhibited B cell transformation by EBV. In fact, 100- to 1,000-fold less tonsilar than peripheral blood NK cells were required to achieve the same protection in vitro, indicating that innate immune control of EBV by NK cells is most efficient at this primary site of EBV infection. The high IFN-γ concentrations, produced by tonsilar NK cells, delayed latent EBV antigen expression, resulting in decreased B cell proliferation during the first week after EBV infection in vitro. These results suggest that NK cell activation by DCs can limit primary EBV infection in tonsils until adaptive immunity establishes immune control of this persistent and oncogenic human pathogen.
Author Summary
Epstein-Barr virus (EBV) establishes a persistent infection in nearly all human adults. Due to its tumor causing potential EBV infection has to be continuously controlled by the immune system in virus carriers. We demonstrate here that in the first week after infection, when other EBV-specific immune responses are still being recruited, human natural killer (NK) cells are able to prevent transformation of the main host cell type by EBV, the human B cell. Especially NK cells of tonsils, the primary site of EBV infection, inhibit B cell transformation by EBV after they have been activated by dendritic cells (DCs). For this protective function, EBV can directly stimulate DCs to efficiently activate NK cells. Interestingly, NK cells primarily prevent B cell transformation by EBV via secretion of the anti-viral cytokine IFN-γ, and NK cells from tonsils and lymph nodes produce 5-fold more of this cytokine than their peripheral blood counterparts. These data suggest that specialized NK cells in tonsils, the mucosal entry site of EBV, can be efficiently stimulated by EBV-activated DCs, and then limit EBV-induced B cell transformation until EBV-specific immune control by other components of the immune system is established.
doi:10.1371/journal.ppat.0040027
PMCID: PMC2233668  PMID: 18266470
19.  Severe chronic active Epstein-Barr virus infection syndrome. 
Clinical Microbiology Reviews  1991;4(1):129-135.
Reports of unusually severe lymphoproliferative disorders associated with extremely high antibody titers against Epstein-Barr virus (EBV) have recently increased. The syndrome, which we designated severe chronic active EBV infection syndrome, is characterized by persistent or intermittent fever, lymphadenopathy, and hepatosplenomegaly and primarily affects children and young adults. Polyclonal gammopathy and bone marrow suppression are generally observed, and some patients develop B-cell or T-cell lymphoproliferation or lymphoma. Frequently, EBV genomes are detectable in tissues infiltrated with lymphoid cells. Additionally, it is difficult to establish spontaneous or B95-8 EBV-induced cell lines despite the expression of an activated EBV infection. We review and report here the published medical literature and our own experience regarding patients with severe chronic active EBV infection syndrome in an attempt to understand this enigmatic syndrome and the possible pathogenetic mechanism(s) responsible for this disorder.
PMCID: PMC358181  PMID: 1848476
20.  A Molecular Link between Malaria and Epstein–Barr Virus Reactivation 
PLoS Pathogens  2007;3(6):e80.
Although malaria and Epstein–Barr (EBV) infection are recognized cofactors in the genesis of endemic Burkitt lymphoma (BL), their relative contribution is not understood. BL, the most common paediatric cancer in equatorial Africa, is a high-grade B cell lymphoma characterized by c-myc translocation. EBV is a ubiquitous B lymphotropic virus that persists in a latent state after primary infection, and in Africa, most children have sero-converted by 3 y of age. Malaria infection profoundly affects the B cell compartment, inducing polyclonal activation and hyper-gammaglobulinemia. We recently identified the cystein-rich inter-domain region 1α (CIDR1α) of the Plasmodium falciparum membrane protein 1 as a polyclonal B cell activator that preferentially activates the memory compartment, where EBV is known to persist. Here, we have addressed the mechanisms of interaction between CIDR1α and EBV in the context of B cells. We show that CIDR1α binds to the EBV-positive B cell line Akata and increases the number of cells switching to the viral lytic cycle as measured by green fluorescent protein (GFP) expression driven by a lytic promoter. The virus production in CIDR1α-exposed cultures was directly proportional to the number of GFP-positive Akata cells (lytic EBV) and to the increased expression of the EBV lytic promoter BZLF1. Furthermore, CIDR1α stimulated the production of EBV in peripheral blood mononuclear cells derived from healthy donors and children with BL. Our results suggest that P. falciparum antigens such as CIDR1α can directly induce EBV reactivation during malaria infection that may increase the risk of BL development for children living in malaria-endemic areas. To our knowledge, this is the first report to show that a microbial protein can drive a latently infected B cell into EBV replication.
Author Summary
Malaria and Epstein–Barr virus (EBV) infections are recognized cofactors in the genesis of endemic Burkitt lymphoma, the most common paediatric cancer in equatorial Africa. EBV is a ubiquitous virus residing in B lymphocytes that establishes a lifelong persistence in the host after primary infection. EBV has two lifestyles: latent infection (non-productive), and lytic replication (productive). Children living in malaria-endemic areas exhibit an elevated viral load, and acute malaria infection increases the levels of circulating EBV. The mechanisms leading to viral reactivation during Plasmodium falciparum malaria infection are not well understood. Cystein-rich inter-domain region 1α (CIDR1α) is a domain of a large protein expressed at the surface of P. falciparum–infected red blood cells. Based on previous findings showing that CIDR1α activates and expands the B cells compartment where EBV persists, we assessed the impact of CIDR1α on viral reactivation. Here, we identify CIDR1α as the first microbial protein able to drive a latently EBV-infected B cell (no virus production) into lytic replication (virus production). Our results suggest that P. falciparum–derived proteins can lead to a direct reactivation of EBV during acute malaria infection, increasing the risk of Burkitt lymphoma development for children living in malaria-endemic areas.
doi:10.1371/journal.ppat.0030080
PMCID: PMC1891325  PMID: 17559303
21.  A Cluster of Virus-Encoded MicroRNAs Accelerates Acute Systemic Epstein-Barr Virus Infection but Does Not Significantly Enhance Virus-Induced Oncogenesis In Vivo 
Journal of Virology  2013;87(10):5437-5446.
Over 90% of the adult human population is chronically infected with the Epstein-Barr virus (EBV), an oncogenic herpesvirus. EBV primarily infects naive human B cells and persists latently in memory B cells. Most individuals experience an asymptomatic infection that is effectively controlled by the adaptive immune response. However, EBV-associated lymphomas can develop in immunocompromised individuals. These tumors typically express all nine EBV latent proteins (latency III). Latency III is also associated with the expression of three precursor microRNAs (miRNAs) located within the EBV BHRF1 gene locus. The role of these BHRF1 miRNAs was unclear until recent in vitro studies demonstrated that they cooperate to enhance virus-induced B cell transformation and decrease the antigenic load of virus-infected cells, indicating that the BHRF1 miRNA cluster may serve as a novel therapeutic target for the treatment of latency III EBV-associated malignancies. However, to date, it is not known if BHRF1 miRNAs enhance virus-induced oncogenesis and/or immune evasion of EBV in vivo. To understand the in vivo contribution of the BHRF1 miRNA cluster to EBV infection and EBV-associated tumorigenesis, we monitored EBV infection and assessed tumor formation in humanized mice exposed to wild-type virus and a viral mutant (Δ123) that lacks all three BHRF1 miRNAs. Our results demonstrate that while the BHRF1 miRNAs facilitate the development of acute systemic EBV infection, they do not enhance the overall oncogenic potential of EBV in vivo.
doi:10.1128/JVI.00281-13
PMCID: PMC3648190  PMID: 23468485
22.  The Viral and Cellular MicroRNA Targetome in Lymphoblastoid Cell Lines 
PLoS Pathogens  2012;8(1):e1002484.
Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus linked to a number of B cell cancers and lymphoproliferative disorders. During latent infection, EBV expresses 25 viral pre-microRNAs (miRNAs) and induces the expression of specific host miRNAs, such as miR-155 and miR-21, which potentially play a role in viral oncogenesis. To date, only a limited number of EBV miRNA targets have been identified; thus, the role of EBV miRNAs in viral pathogenesis and/or lymphomagenesis is not well defined. Here, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) combined with deep sequencing and computational analysis to comprehensively examine the viral and cellular miRNA targetome in EBV strain B95-8-infected lymphoblastoid cell lines (LCLs). We identified 7,827 miRNA-interaction sites in 3,492 cellular 3′UTRs. 531 of these sites contained seed matches to viral miRNAs. 24 PAR-CLIP-identified miRNA:3′UTR interactions were confirmed by reporter assays. Our results reveal that EBV miRNAs predominantly target cellular transcripts during latent infection, thereby manipulating the host environment. Furthermore, targets of EBV miRNAs are involved in multiple cellular processes that are directly relevant to viral infection, including innate immunity, cell survival, and cell proliferation. Finally, we present evidence that myc-regulated host miRNAs from the miR-17/92 cluster can regulate latent viral gene expression. This comprehensive survey of the miRNA targetome in EBV-infected B cells represents a key step towards defining the functions of EBV-encoded miRNAs, and potentially, identifying novel therapeutic targets for EBV-associated malignancies.
Author Summary
Over 90% of adults worldwide are infected with Epstein-Barr virus (EBV). While EBV infection is normally controlled by a healthy immune system, in immuno-compromised individuals, EBV can cause serious disease and/or cancer. During infection, EBV expresses viral microRNAs (miRNAs) and induces the expression of specific cellular miRNAs. In general, miRNAs inhibit target gene expression by binding to complementary regions on target messenger RNAs (mRNA). While cellular miRNAs regulate important biological processes such as cell growth and differentiation, and many miRNAs have been linked to cancer progression, the functions of EBV miRNAs are largely unknown. To identify targets of EBV miRNAs and cellular miRNAs in EBV-infected cells, we used a high-throughput method based on next-generation sequencing technology to give a global picture of miRNA-regulated gene expression. Our analysis showed that over 500 mRNAs can be regulated by viral miRNAs, many of which are directly relevant to EBV infection. This study provides a comprehensive survey of viral and cellular miRNA targets in B cells, which is a positive step towards identifying novel therapeutic targets for EBV-associated cancers.
doi:10.1371/journal.ppat.1002484
PMCID: PMC3266933  PMID: 22291592
23.  Assessment of immunological changes in Epstein-Barr virus co-infection in Egyptian chronic HCV patients 
Memórias do Instituto Oswaldo Cruz  2014;109(6):722-727.
Epstein-Barr virus (EBV) plays a major role in liver pathology. Similar to other members of the herpesvirus family, EBV establishes a persistent infection in more than 90% of adults. The aim of this study was to evaluate the impact of EBV and chronic hepatitis C co-infection (HCV) on biochemical and immunological responses in patients. The study was conducted in 62 patients and 33 apparently healthy controls. Patients were divided into three groups: group I, consisting of 31 patients with chronic hepatitis C infection (CHC), group II, consisting of eight patients with EBV infection and without HCV infection and group III, consisting of 23 patients with EBV and chronic HCV. The percentage of CD3+ cells, helper CD4+ cells and CD19+ B-cells was measured by flow cytometry. Human interferon-γ (IFN-γ) and interleukin (IL)-15 levels were measured by an ELISA. The levels of liver alanine aminotransferase and aspartate aminotransferase enzymes were higher in EBV/HCV patients compared to that in EBV and HCV mono-infected patients. EBV/HCV patients had significantly reduced percentages of CD3+ and CD4+ cells compared to EBV patients. Serum IFN-γ levels were significantly reduced in EBV/HCV patients (3.86 pg/mL) compared to CHC patients (6.76 pg/mL) and normal controls (4.69 pg/mL). A significant increase in serum IL-15 levels was observed in EBV/HCV patients (67.7 pg/mL) compared to EBV patients (29.3 pg/mL). Taken together, these observations suggest that HCV and EBV co-infection can potentiate immune response dampening in patients.
doi:10.1590/0074-0276140049
PMCID: PMC4238763  PMID: 25317700
Epstein-Barr virus; hepatitis C virus; IFN-γ; IL-15
24.  EBV in Hodgkin Lymphoma 
Up to 40% of Hodgkin lymphoma (HL) cases are associated with the Epstein-Barr virus (EBV). Clonal viral genomes can be found in the HL tumor cells, the Hodgkin Reed-Sternberg cells (HRS). The latent infection results in expression of the viral oncogenes LMP1 and LMP2A which contribute to generate the particular phenotype of the HRS cells. EBV does not only undergo epigenetic changes of its genome during latency, but also induces epigenetic changes in the host genome. The presence of EBV may alter the composition and activity of the immune cells surrounding the HRS cells. EBV favours a Th1 reaction, but this attempt at a cell mediated immune response appears to be ineffective. The presence of EBV in HL is associated with several clinicopathological characteristics: It is more frequent in cases with mixed cellular histology, in males, in children and older adults, and in developing countries, while the young-adult onset HL of nodular sclerosis type in industrialized countries is typically EBV-negative. Countries in the Mediterranean area often show an intermediate epidemiological pattern. Recent studies suggest a genetic predisposition to develop EBV-associated HL. Circulating EBV-DNA may serve as a biomarker to monitor response to therapy, and eventually, EBV will become a target for therapeutic intervention also in HL.
doi:10.4084/MJHID.2009.013
PMCID: PMC3033177  PMID: 21416003
25.  Haemophagocytic syndrome and elevated EBV load as initial manifestation of Hodgkin lymphoma in a HIV patient: case report and review of the literature 
Journal of the International AIDS Society  2014;17(4Suppl 3):19650.
Introduction
In HIV patients, haemophagocytic syndrome (HPS) may occur in the presence of cancer, concomitant viral infection, HIV primo-infection or at the initiation of highly active antiretroviral therapy (HAART). Hodgkin lymphoma remains a rare cause of HPS. We describe a case of HPS with very high Epstein Barr virus (EBV) load in a HIV patient as initial manifestation of Hodgkin lymphoma.
Materials and Methods
A 29-year-old HIV positive man, successfully treated with HAART with an undetectable viral load and CD4 cells count of 438/µl, was admitted for high fever of unknown origin. Laboratory results showed a pancytopenia with haemoglobin at 82 g/l, lymphocyte count at 0.36G/l and platelets count at 47G/l; a highly elevated ferritine >7500 µg/l; increased lactate dehydrogenase at 885U/l and soluble IL2 receptor (CD25) >60 ng/ml. EBV load was measured and confirmed at 2,600,000 copies/ml. A PET-CT imaging showed diffuse elevated metabolic activity in the bone marrow and in two lesions in the spleen without lymphadenopathy. Bone marrow and liver biopsies revealed images of haemophagocytosis and lymphocyte depleted Hodgkin lymphoma. Treatment consisted in etoposid, steroids, and R-ABVD (rituximab, doxorubicin, bleomycin, vinblastine, dacarbazine) chemotherapy. The patient completed six cycles of chemotherapy. We reviewed the literature in PubMed with the following keywords: HPS, HIV, EBV, Hodgkin lymphoma.
Results
We identified four publications and two reviews reporting cases of HPS associated with Hodgkin lymphoma in HIV patients with either a positive EBV load either the presence of encoded EBV RNA in tumour cells. Twenty-two cases (including one pediatric case) were described. Among adults, mostly men, the median age was <50 years and immune suppression was marked with a median CD4 cell count<100 cells/µl, even in patients receiving HAART. When measured, EBV load in the serum was high. Prognosis was poor with a high mortality despite adequate treatment consisting in steroids and chemotherapy, with or without etoposide (Table 1).
Conclusions
Our case report and the review of literature suggest that physicians should be aware of the association between EBV infection/reactivation and Hodgkin lymphoma as a cause of HPS in HIV patients, even if successfully treated with HAART. The pathogenesis of these three interrelated conditions (viral infection, oncogenesis and immunologic reaction) remains unclear.
doi:10.7448/IAS.17.4.19650
PMCID: PMC4224866  PMID: 25394154

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