Increased cell-free viral DNA in fatal cases of chronic active Epstein-Barr virus infection.

We have studied the nature of Epstein-Barr virus (EBV) infection in 33 patients with chronic active EBV infection. The study population included 14 patients with fatal chronic EBV infection and 19 patients with nonfatal chronic EBV infection, as well as 18 patients with acute EBV-induced infectious mononucleosis and 10 healthy controls. EBV DNA was measured in serum or plasma samples from the patients by semiquantitative polymerase chain reaction-based assay. EBV DNA was detected in serum or plasma samples from 62% (9/14) of patients with fatal chronic active EBV infection. In contrast, only 11% (2/19) of patients with nonfatal chronic active EBV infection and 11% (2/18) of patients with infectious mononucleosis displayed EBV DNA. None of the healthy controls tested positive. Cell-free circulating EBV DNA may represent an important feature of chronic active EBV infection and may provide a useful tool to monitor the severity of this illness.     

Biology of Epstein-Barr virus during infectious mononucleosis

Infectious mononucleosis is the clinical manifestation of primary infection with Epstein-Barr virus (EBV). We monitored primary infection during convalescence and during the establishment of persistent infection. The profiles of EBV strains in the oral cavity and in peripheral blood were determined by use of a heteroduplex tracking assay specific for the EBV gene encoding latent membrane protein 1. Multiple EBV strains were detected in most patients and persisted in and were possibly transmitted among 3 distinct compartments of infection, including the oral cavity, peripheral blood lymphocytes, and the cell-free fraction of the blood plasma. We also tracked transmission of multiple strains from an asymptomatic carrier to a patient diagnosed with primary EBV infection. These data reveal that primary EBV infection is complex, with transmission of multiple strains and clear differences in relative abundance of strains in distinct compartments.     

Simultaneous infection with multiple herpesviruses.

Active dual infection with Epstein-Barr virus (EBV) and cytomegalovirus (CMV) was observed in four otherwise healthy persons with mononucleosis syndromes. A secondary serologic response to EBV occurred in three patients as determined by the presence of antibodies to EBV-induced nuclear antigen (EBNA) early in the illness. All four patients lacked heterophil antibodies; in the one case tested, immunoglobulin M (IgM) antibodies specific for EBV viral capsid antigen (VCA) were absent as well. The Guillain-Barré syndrome occurred in one patient, who also had active infection with herpes simplex virus 1 (HSV-1). In a fifth patient, herpes zoster developed complicating heterophil-positive infectious mononucleosis due to primary infection with EBV.These five cases demonstrate that mononucleosis syndromes may occur in association with dual or multiple herpesvirus infections and that reactivation of EBV may be common during heterophil-negative mononucleosis. Reactivation of latent virus is most likely related to depressed cellular immunity due to a primary infection with another herpesvirus. An alternate hypothesis is that viral DNA polymerase induced by infection with one herpesvirus might simultaneously permit the productive replication of a second herpesvirus previously latent within the same cell. Thus, reactivation may result from molecular interactions between viruses at the cellular level.The possibility of multiple infections must be considered whenever determining the specific viral etiology of heterophil-negative mononucleosis.     

Lymphocyte reactivity in infectious mononucleosis

Reactivity of lymphocytes to a purified preparation of Epstein-Barr virus (EBV) was studied in 17 healthy individuals and 15 patients with primary EVB infection and clinical signs of infectious mononucleosis. Lymphocyte reactivity to EBV was negative in individuals who were seronegative for antibody to EBV and in seven of 15 patients examined less than or equal to 21 days after onset of clinical signs of illness. Positive lymphocyte reactivity was observed in all patients by day 36; once it was established, it remained in individual patients for up to 480 days. During the acute phase of infectious mononucleosis, negative lymphocyte reactivity was always associated with a strong antibody response to EBV capsid antigens. This disparity was paralleled by the inability of lymphocytes to respond to recall antigens and mitogens, especially concanavalin A. Positive lymphocyte reactivity to EBV indicates a specific cellular memory function, probably of thymus-cell origin, which is acquired following primary EBV infection, and may be retained into later life.     

Malignant granular lymphoproliferation after Epstein-Barr virus infection: partial immunologic reconstitution with interleukin-2

This report describes a patient who developed a malignant proliferation of granular lymphocytes following Epstein-Barr virus (EBV) infection. For many months, his illness resembled prolonged infectious mononucleosis with persistent fatigue, fever, leukocytosis, and serologic evidence of recent primary EBV infection. After approximately 1 year, however, he developed progressive granular lymphocytosis and extensive lymphocytic infiltration of the bone marrow and liver. Tests for EBV DNA in pre- and postmortem tissue samples using a sensitive DNA hybridization technique were negative. Southern blot analysis of DNA prepared from blood mononuclear cells demonstrated clonal T-cell antigen receptor gene rearrangement. Despite increased numbers of circulating lymphocytes with the morphology and surface phenotype of normal donor natural killer (NK) cells, the patient's NK activity was consistently depressed in a standard in vitro assay. However, in vitro incubation with interleukin-2 (IL-2), but not with alpha- or gamma-interferon, increased the NK activity of the patient's lymphocytes. Intravenous recombinant IL-2 treatment transiently increased the patient's blood NK activity and was associated with seroconversion to EBV nuclear antigens but failed to affect the progression of his disease. Our findings indicate that clonal granular lymphocytic proliferation may develop after EBV infection and confirm the utility of DNA hybridization analysis in distinguishing monoclonal from benign immunoreactive lymphoproliferation. Furthermore, our results suggest that certain functionally inert neoplastic granular lymphocytes acquire NK activity when exposed to IL-2.     

A boy with fatal infectious mononucleosis suspected as the first Japanese case of X-linked lymphoproliferative syndrome

We report a case of a 10-month-old boy who died of severe hepatic failure after a prolonged course of infectious mononucleosis. He also presented interstitial pneumonitis, meningoencephalitis and aplastic anaemia. Epstein-Barr virus (EBV)-specific cytotoxic T lymphocyte (CTL) activity had not been detected in his peripheral blood during the course of the illness. Studies of his mother revealed a severe reactivation pattern of anti-EBV antibodies and decreased EBV-specific CTL activity. An X-linked familial susceptibility to EBV infection such as X-linked lymphoproliferative syndrome (XLP) might be associated with his fatal EBV infection.     

Asymptomatic primary Epstein-Barr virus infection occurs in the absence of blood T-cell repertoire perturbations despite high levels of systemic viral load.

Primary infection with the human herpesvirus, Epstein-Barr virus (EBV), may result in subclinical seroconversion or may appear as infectious mononucleosis (IM), a lymphoproliferative disease of variable severity. Why primary infection manifests differently between patients is unknown, and, given the difficulties in identifying donors undergoing silent seroconversion, little information has been reported. However, a longstanding assumption has been held that IM represents an exaggerated form of the virologic and immunologic events of asymptomatic infection. T-cell receptor (TCR) repertoires of a unique cohort of subclinically infected patients undergoing silent infection were studied, and the results highlight a fundamental difference between the 2 forms of infection. In contrast to the massive T-cell expansions mobilized during the acute symptomatic phase of IM, asymptomatic donors largely maintain homeostatic T-cell control and peripheral blood repertoire diversity. This disparity cannot simply be linked to severity or spread of the infection because high levels of EBV DNA were found in the blood from both types of acute infection. The results suggest that large expansions of T cells within the blood during IM may not always be associated with the control of primary EBV infection and that they may represent an overreaction that exacerbates disease.     

Serum levels of lymphokines and soluble cellular receptors in primary Epstein-Barr virus infection and in patients with chronic fatigue syndrome.

The immunopathology in primary Epstein-Barr virus (EBV) infections and in chronic fatigue syndrome was studied by examining serum levels of interleukins (IL) and of soluble T cell receptors in serum samples. Serum samples were from patients during and 6 months after primary EBV-induced infectious mononucleosis and from patients with chronic fatigue syndrome and serologic evidence of EBV reactivation. Markers for T lymphocyte activation (soluble IL-2 and CD8) and for monocyte activation (neopterin) were significantly elevated during acute infectious mononucleosis but not in patients with chronic fatigue syndrome. Interferon-alpha, IL-1 beta, and IL-6 levels were not significantly increased in any patient group but inferferon-gamma levels were significantly increased during the acute phase of infectious mononucleosis. The levels of IL-1 alpha were significantly higher than in controls both in patients with infectious mononucleosis and in those with chronic fatigue syndrome. In the latter, the lack of most markers for lymphocyte activation found in patients with infectious mononucleosis makes it less likely that EBV reactivation causes symptoms.     

Epstein-Barr virus and the elderly host

The ability of the Epstein-Barr virus (EBV) to cause latent lifelong infection in the host and its capabilities of transformation may have important implications for the elderly host. Reports in the literature and hospital records were reviewed to determine the activity of EBV in the elderly. Seroepidemiologic surveys demonstrated that 90%-97% of adults more than 60 years old were seropositive for EBV. Geometric mean antibody titers and the percentage of individuals with high antibody titers to EBV increased with age--changes that were not associated with clinical illness. Only 29 cases of infectious mononucleosis have been reported in adults more than 60 years old. The elderly with infectious mononucleosis had significantly fewer occurrences of pharyngitis, lymphadenopathy, and splenomegaly when compared with young adults. The cases of two patients with illnesses that did not meet full criteria for infectious mononucleosis but may still have represented clinical manifestations of EBV infection are presented. Other EBV-associated diseases reported in the elderly include nasopharyngeal carcinoma and possibly B cell lymphoproliferative disease but not a chronic mononucleosis-like syndrome.     

Multiple Epstein-Barr virus strains in patients with infectious mononucleosis: comparison of ex vivo samples with in vitro isolates by use of heteroduplex tracking assays

Recent work using a heteroduplex tracking assay (HTA) to identify resident viral sequences has suggested that patients with infectious mononucleosis (IM) who are undergoing primary Epstein-Barr virus (EBV) infection frequently harbor different EBV strains. Here, we examine samples from patients with IM by use of a new Epstein-Barr nuclear antigen 2 HTA alongside the established latent membrane protein 1 HTA. Coresident allelic sequences were detected in ex vivo blood and throat wash samples from 13 of 14 patients with IM; most patients carried 2 or more type 1 strains, 1 patient carried 2 type 2 strains, and 1 patient carried both virus types. In contrast, coresident strains were detected in only 2 of 14 patients by in vitro B cell transformation, despite screening >20 isolates/patient. We infer that coacquisition of multiple strains is common in patients with IM, although only 1 strain tends to be rescued in vitro; whether nonrescued strains are present in low abundance or are transformation defective remains to be determined.     

Persistently high Epstein-Barr virus (EBV) loads in peripheral blood lymphocytes from patients with chronic active EBV infection

Chronic active Epstein-Barr virus infection (CAEBV) is a severe illness with unusual EBV activation that persists for years, and its pathogenesis is largely unknown. After the creation of an accurate and reproducible polymerase chain reaction system to quantify EBV DNA, virus loads in peripheral blood lymphocytes (PBL) were determined in 54 children: 15 with CAEBV, 16 with infectious mononucleosis (IM), and 23 healthy children. Children with CAEBV and those with IM had high virus loads. Lower loads were detected in 47% of seropositive healthy donors. There were two distinct differences between children with CAEBV and those with IM: The former had greater viral replication (10(3)-10(7) copies/2.5x10(5) PBL) than those with IM, and viral replication declined in children with IM whereas active replication persisted for years in subjects with CAEBV. Persisting high virus loads are a possible diagnostic criterion for CAEBV. EBV loads may enable classification and prognosis of EBV infections.     

Treatment of primary Epstein-Barr virus infection in patients with X-linked lymphoproliferative disease using B-cell-directed therapy

X-linked lymphoproliferative disease (XLP) is a congenital immunodeficiency that is characterized by an abnormal immune response to primary Epstein-Barr virus (EBV) infection. After EBV exposure, affected patients often develop fulminant infectious mononucleosis (FIM), a life-threatening condition marked by the uncontrolled expansion and activation of T and B lymphocytes and macrophages. We hypothesized that the rapid elimination of B cells immediately following EBV exposure might reduce the severity of primary EBV infection in patients with XLP. To test this possibility, we administered the anti-CD20 antibody rituximab to 2 patients who presented with acute infection. Following treatment, both patients exhibited a complete resolution of symptoms and no longer demonstrated detectable EBV DNA within circulating lymphocytes. Moreover, neither patient has developed FIM or lymphoma in more than 2 years of follow-up. These data suggest that the pre-emptive use of B-cell-directed therapy may reduce the morbidity and mortality of primary EBV infection in XLP-affected individuals.     

Persistent transfusion-associated infectious mononucleosis with transient acquired immunodeficiency

Trauma and blood transfusion led to profound, persistent infectious mononucleosis in a 21 year old man. Splenectomy and trauma had apparently produced transient immune deficiency which was complicated by osteomyelitis of a fractured tibia. The transfused blood probably contained Epstein-Barr virus (EBV). Infectious mononucleosis had ensued 25 days after a blood transfusion was given, antibodies to EBV appeared in his serum, and the infectious mononucleosis persisted for nearly two years. His immunity returned gradually to normal, but because of nonunion of the fracture site, which was infected by Staphylococcus aureus, above-knee amputation was required. The acquired, transient immune deficiency to EBV and profound infectious mononucleosis seen in this patient is analogous to inherited, permanent immune deficiency to this virus in the X-linked lymphoproliferative syndrome.     

Prolonged illness after infectious mononucleosis is associated with altered immunity but not with increased viral load

BACKGROUND: Primary Epstein-Barr virus (EBV) infection causes a spectrum of characteristics that range from asymptomatic seroconversion to severe infectious mononucleosis (IM), sometimes with prolonged symptoms and disability. We examined the relationships between clinical course, number of viral copies, and immunological parameters in a prospective cohort of subjects with recent IM. METHODS: Eight case patients with at least 6 months of disabling symptoms and 31 matched control subjects who had recovered promptly were included. Symptom scores were recorded at regular intervals over the course of 12 months. Cellular EBV load, EBV-specific antibody responses, lymphocyte subsets, and EBV-specific interferon (IFN)- gamma induction were measured. RESULTS: In case patients with prolonged illness, the severity of acute-phase symptoms was greater, the development of anti-EBV nuclear antigen-1 immunoglobulin G was more rapid, and the time to development of the peak IFN- gamma response to the majority of latent-cycle EBV peptides was generally slower than those in control subjects. However, in both groups, neither viral nor immune parameters correlated with the severity or duration of symptoms. CONCLUSIONS: The resolution of symptomatic IM is not determined by control of viremia, nor is it easily explained by altered host responses to EBV infection. The detailed determinants of delayed recovery remain to be elucidated.     

Heterophil-negative infectious mononucleosis and mononucleosis-like illnesses. Laboratory confirmation of 43 cases.

During a 50-month period the diagnosis of heterophil antibody negative infectious mononucleosis or of a mononucleosis-like illness was made in 43 patients with a variable clinical picture and significant numbers of atypical lymphocytes. Epstein-Barr virus (EBV)-related serologic tests revealed that seven patients had primary EBV infections based on the detection of immunoglobulin M (IgM) antibodies to EB-viral capsid antigens (IgM-VCA) and the absence of anti-Epstein-Barr virus associated nuclear antigen (EBNA) on most initial specimens (six of seven cases). Thirty cases were due to active cytomegalovirus (CMV) infections and both detectable CMV-macroglobulins (≧1:32) and significant anti-CMV titers were present by a complement fixation technic. Abnormalities in liver function were less marked in CMV than in EBV infections in age-matched subjects. Of the remaining six cases, one was due to rubella and one to toxoplasmosis. Four cases were of undetermined etiology. Serums from 38.1 per cent of the patients with heterophil-antibody positive infectious mononucleosis were found to “cross react” in the IgM-CMV test, but serums from patients with acute CMV infection did not cross react in the VCA-specific IgM test. In nine of 36 cases without heterophil antibody (six due to CMV, one due to toxoplasmosis and one apparent infectious hepatitis), anti-D or -R of the early-antigen (EA) complex was detected (1:10 to 1:40), raising the question of reactivation of the EBV-carrier state by intervening infections mainly of viral origin.     

Epstein-Barr virus induced hepatitis: An important cause of cholestasis.

INTRODUCTION:: Epstein-Barr virus (EBV) infection frequently involves the liver, presenting as elevations in transaminases. EBV infection associated hepatitis, presenting with hyperbilirubinemia is rare. We describe a case of infectious mononucleosis that presented with cholestatasis, and summarize 23 cases from the literature to categorize this increasingly recognized clinical spectrum of EBV infection induced cholestatic hepatitis. METHODS:: We conducted an extensive literature review of all cases of EBV in pediatric and adult literature with cholestatasis using MEDLINE and EMBASE. We also included information on one case from our institution. RESULTS:: We identified 24 cases. Median age was 20 years (range 1-72 years), with 14 (58%) females. On presentation, fever (72%), jaundice (67%) and splenomegaly (62%) were the most common signs. Laboratory data revealed the median asparate aminotransferase (AST), or alanine aminotransferase (ALT) level was 179IU/L (range 56-2518IU/L), median serum bilirubin level 12.6mg/dL (range 2.2-47.5mg/dL) and median alkaline phosphatase level 749IU/L (range 31-3105IU/L). Diagnosis was confirmed using EBV viral capsid antigen IgM in 20 (83%) patients. HIV testing was done in 7 (29%) of the cases, and was negative. One patient died from the illness, while full recovery was reported in all other cases, with median follow-up of 30 days (range 5-180 days). CONCLUSIONS:: Cholestatasis is associated with EBV infection, and should be part of the differential diagnosis in all age groups, presenting with hyperbilirubinemia.     

DNA-DNA dot hybridization to detect Epstein-Barr virus in throat washings

To compare the abilities of the nucleic acid dot hybridization assay and the cord blood lymphocyte transformation assay to detect Epstein-Barr virus (EBV), we examined throat washings from healthy control subjects (nine EBV-seronegative and 51 EBV-seropositive), patients with acute infectious mononucleosis, and renal transplant recipients. The dot hybridization assay detected EBV excretion in four (8%) of the EBV-seropositive controls; three of these four were also positive by the lymphocyte transformation assay. Throat washings from seven (87.5%) of eight patients with acute infectious mononucleosis were positive by both assays. EBV was present in throat washings from 13 (50%) of 26 renal transplant recipients. For specimens stored at -70 C for less than four months, the dot hybridization assay had a sensitivity of 90% and a specificity of 98% when compared with the lymphocyte transformation assay. The dot hybridization assay is a rapid, sensitive, and specific test that can be performed on readily available clinical specimens.     

Recombinant gp350 vaccine for infectious mononucleosis: a phase 2, randomized, double-blind, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of an Epstein-Barr virus vaccine in healthy young adults

BACKGROUND: To date, there is no commercially available vaccine to prevent infectious mononucleosis, a disease frequently induced by Epstein-Barr virus (EBV) infection in adolescents or adults devoid of preexisting immunity to the virus. METHODS: A total of 181 EBV-seronegative, healthy, young adult volunteers were randomized in a double-blind fashion to receive either placebo or a recombinant EBV subunit glycoprotein 350 (gp350)/aluminum hydroxide and 3-O-desacyl-4'-monophosphoryl lipid A (AS04) candidate vaccine in a 3-dose regimen. RESULTS: The vaccine had demonstrable efficacy (mean efficacy rate, 78.0% [95% confidence interval {CI}, 1.0%-96.0%]) in preventing the development of infectious mononucleosis induced by EBV infection, but it had no efficacy in preventing asymptomatic EBV infection. One month after receipt of the final dose of gp350 vaccine, 98.7% of subjects showed seroconversion to anti-gp350 antibodies (95% CI, 85.5%-97.9%), and they remained anti-gp350 antibody positive for >18 months. Furthermore, there were no concerns regarding the safety or reactogenicity of the gp350/AS04 vaccine. CONCLUSION: These data support the clinical feasibility of using an EBV vaccine to prevent infectious mononucleosis. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00430534.     

Leukemia-like syndrome in Balb/c mice infected with the lymphotropic gamma herpesvirus MHV-Sumava: an analogy to EBV infection

Based on our previous observation that primary infection with the murine gamma herpesvirus (MHV) isolate Sumava (MHV-SU) undergoes a lymphoproliferative phase resembling to Epstein-Barr virus (EBV) induced infectious mononucleosis (IM), we evaluated white blood cell (WBC) counts at late stages following MHV-SU infection. In consequence of intranasal inoculation with MHV-SU a leukemia-like syndrome in Balb/c mice developed. The syndrome in question was accompanied with significant splenomegaly; in the peripheral blood leukocytosis (from 8 x 10(4) to 5 x 10(5) leukocytes/microl) and a high percentage of atypical lymphocytes (60-80%) was found. Presented results are bringing further evidence for lymphoproliferative effect of MHV and point at analogic course of MHV-SU and EBV infections.