Development of real-time PCR assays for the quantitative detection of Epstein-Barr virus and cytomegalovirus,comparison of TaqMan probes,and molecular beacons

Human Epstein-Barr virus (EBV) and cytomegalovirus (CMV) can cause serious complications in immunocompromised patients. Rapid diagnosis of EBV and CMV infection is critical in the management of the disease so that anti-viral therapy can be started early. Here we describe the development of real-time PCR assays using TaqMan probes and molecular beacons and compare the performance of both assays with a well-established, validated, gel-based PCR method for the quantification of EBV and CMV in patients’ samples. The TaqMan and molecular beacon assays were linear between 10 to 10⁷ viral genomes/reaction. Both assays generated calibration curves with strong correlation and low intra-assay and interassay variation. Results of EBV and CMV viral load determination inpatient samples obtained by the gel-based and real-time PCR were very similar. The real-time PCR assays showed increases in viral load before clinical measures of viral disease and decreases in viral load during anti-viral therapy in two of six pediatric patients. The data indicate that these TaqMan and molecular beacon approaches are accurate, rapid, and reliable assays for the diagnosis and monitoring of EBV and CMV infections in patients.     

Decreased IL-7 signaling in T cells from patients with PTLD after allogeneic HSCT

Interleukin (IL)-7, a nonredundant cytokine for B and T cells, plays a central role in cell survival and immune memory formation. Peripheral blood mononuclear cells (PBMCs) from 7 patients after hematopoietic stem cell transplantation (HSCT) diagnosed with posttransplantation lymphoproliferative disease (PTLD) and from 10 Epstein-Barr virus (EBV) polymerase chain reaction-positive HSCT patients (controls) were evaluated for IL-7- and IL-2 induced Stat5 phosphorylation in CD4+ and CD8+ T cells. PBMCs from PTLD+ and control patients exhibited detectable EBV specific CD8+ T cells defined by tetramer analysis. CD4+ and CD8+ T cells from patients with PTLD showed statistically significant reduction in responsiveness to IL-7 compared with PBMCs obtained from controls defined by Stat5 phosphorylation. CD20+ B cells from patients with PTLD and from some EBV+ polymerase chain reaction control individuals exhibited IL-7R expression. Dysregulated immune surveillance, reflected by deficient Stat5 phosphorylation, may facilitate PTLD development despite the presence of EBV-reactive CD8+ T cells. Reduced IL-7 responsiveness will aid to monitor patients after HSCT for increased risk to develop EBV-associated PTLD.     

Epstein-barr virus-infected resting memory B cells, not proliferating lymphoblasts, accumulate in the peripheral blood of immunosuppressed patients.

When Epstein-Barr virus (EBV) infects B cells in vitro, the result is a proliferating lymphoblast that expresses at least nine latent proteins. It is generally believed that these cells are rigorously controlled in vivo by cytotoxic T cells. Consistent with this, the latently infected cells in the peripheral blood of healthy carriers are not lymphoblasts. Rather, they are resting memory B cells that are probably not subject to direct immunosurveillance by cytotoxic T lymphocytes (CTLs). When patients become immunosuppressed, the viral load increases in the peripheral blood. The expansion of proliferating lymphoblasts due to the suppressed CTL response is believed to account for this increase and is considered to be a major risk factor for posttransplant lymphoproliferative disease (PTLD) and AIDS-associated B cell lymphoma. Here we show that there is an increase in the numbers of latently infected cells in the peripheral blood of immunosuppressed patients. However, the cells are not proliferating lymphoblasts. They are all latently infected, resting, memory B cells-the same population of infected cells found in the blood of healthy carriers. These results are discussed in the context of a model for EBV persistence that explains why PTLD is usually limited to the lymph nodes.     

Predictive value of quantitative PCR-based viral burden analysis for eight human herpesviruses in pediatric solid organ transplant patients

Human herpesviruses can cause significant morbidity and mortality in pediatric solid organ transplant recipients. It was hypothesized that viral burden quantification by polymerase chain reaction using an internal calibration standard could aid in distinguishing between viral disease and latency. Here we report the results of a 2-year prospective study of 27 pediatric solid organ (liver, kidney, or heart) transplant recipients in which multiple samples were analyzed for levels of all eight human herpesviruses by internal calibration standard-polymerase chain reaction. Herpes simplex viruses 1 and 2, varicella-zoster virus, and Kaposi’s sarcoma-associated herpesvirus were not detected in any of these samples. Human herpesvirus types 6 and 7 were detected in half of the patients, but were present at low levels, similar to those found in reference populations. Epstein-Barr virus (EBV) and cytomegalovirus (CMV) were detected in 89% and 56% of the patients, respectively. Viral burden analysis suggested distinct patient populations for CMV, with a natural cutoff of 10,000 viral targets/ml blood strongly associated with disease. In some cases, a dramatic increase in CMV levels preceded clinical evidence of disease by several weeks. EBV viral burden was relatively high in the only patient presenting with an EBV syndrome. However, two other patients without evidence of EBV disease had single samples with high EBV burden. Rapid reduction in both EBV and CMV burden occurred with antiviral treatment. These data suggest that viral burden analysis using internal calibration standard-polymerase chain reaction for CMV, and possibly other herpesviruses, is an effective method for monitoring pediatric transplant patients for significant herpesvirus infection and response to therapy.     

Validation of Roche LightCycler Epstein-Barr virus quantification reagents in a clinical laboratory setting

Epstein-Barr virus (EBV) is associated with a wide range of benign and malignant diseases, including infectious mononucleosis, lymphoma, posttransplant lymphoproliferative disorder, and nasopharyngeal carcinoma. Measurement of EBV viral load in plasma is increasingly used for rapid assessment of disease status. We evaluated the performance characteristics of an EBV polymerase chain reaction assay that uses commercial reagents and instruments from Roche Diagnostics (Indianapolis, IN). DNA was extracted from plasma using a MagNaPure instrument, and viral load was measured by real-time polymerase chain reaction on a LightCycler. Analyte-specific reagents included primers and hybridization probes targeting the EBV LMP2 gene and a spiked control sequence. Accuracy and reproducibility were established using DNA from three cell lines. The assay was sensitive to approximately 750 copies of EBV DNA per milliliter of plasma and was linear across at least four orders of magnitude. The assay detected EBV DNA in three of five samples from nasopharyngeal carcinoma patients, seven of nine infectious mononucleosis samples, and 34/34 samples from immunosuppressed patients with clinically significant EBV-related disease, whereas EBV DNA was undetectable in plasma from 21 individuals without EBV-related disease. In conclusion, this LightCycler EBV assay is rapid, sensitive, and linear for quantifying EBV viral load. The assay appears to be useful for measuring clinically significant EBV levels in immunodeficient patients.     

Efficacy of Epstein-Barr virus removal by leukoreduction of red blood cells

BACKGROUND: Epstein-Barr virus (EBV) infection results in life-long carriage of latent virus in B lymphocytes in the majority of the adult population, including blood donors. The removal of EBV from red blood cell (RBC) components by leukoreduction was assessed. STUDY DESIGN AND METHODS: Sixteen randomly selected fresh AS-5 units were leukoreduced by filtration. B lymphocytes from preleukoreduction specimens and mononuclear cells (MNCs) from postleukoreduction specimens were assayed for EBV DNA with sensitive real-time polymerase chain reaction (PCR). RESULTS: EBV genomes were detected in CD19+ B cells in 14 of 16 preleukoreduced RBC units. EBV genomic copy number in the units ranged from 0.18 to 96.84 per 10(5) B lymphocytes representing approximately 135 to 72,630 total EBV genomes per bag. Leukoreduction rendered all but one unit EBV-negative by PCR. The lone PCR-positive unit after leukoreduction amplified 1.2 EBV genome copies from MNCs recovered from the entire unit of leukoreduced RBCs; this unit had the highest EBV viral load before leukoreduction (72,630 EBV genomes). CONCLUSIONS: These results indicate that a 4-log reduction of EBV genomic copy number can be achieved with leukoreduction of RBC units and renders most RBC units EBV-negative by sensitive PCR.     

Quantification of epstein-barr virus DNA is helpful for evaluation of chronic active epstein-barr virus infection

Chronic active Epstein-Barr virus infection (CAEBV) presents with chronic or recurrent infectious mononucleosis-like symptoms, such as low-grade fever, liver dysfunction, lymphadenopathy, and hepatosplenomegaly. Immunological methods are useful for the diagnosis of viral infections. However, CAEBV patients do not necessarily have high titers of Epstein-Barr virus (EBV)-specific antibodies. Hosts that are immunocompromised after hematopoietic stem cell transplantations sometimes suffer from systemic EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and EBV-positive lymphoma. Patients with EBV-associated diseases are often diagnosed by analyses of bone marrow. Cytomegalovirus (CMV) can cause serious pneumonia or retinitis in immunocompromised hosts. In order to noninvasively understand the clinical status of patients with EBV-associated diseases, we conducted real-time polymerase chain reaction (PCR) methods in their peripheral blood in order to quantify EBV and CMV DNA levels, which reflect viral activity. Here, we describe a 30-year-old Japanese female patient with CAEBV. The patient had repeated fever, fatigue, and liver dysfunction. The histopathological results of liver biopsies were positive for EBV-encoded RNA-1. Acute hepatitis was associated with the EBV infection. The whole-blood EBV DNA levels were high and above 1.0 × 10⁷ copies/mL. After immunosuppressive and antiviral therapies, EBV DNA levels lowered. However, she had to receive bone marrow transplantation because of her EBV-HLH. As the number of lymphocytes increased in the post-transplantation period, EBV DNA levels gradually increased again. The simultaneous detection of CMV DNA was more sensitive than the CMV antigenemia test that is often used to diagnose CMV infections. Unfortunately, the patient died due to a fungal infection. Observing EBV DNA levels closely with real-time quantitative PCR methods is helpful for evaluating the changes in the clinical course.     

异基因造血干细胞移植后Epstein-Barr病毒相关性中枢神经系统移植后淋巴增殖性疾病诊治的研究进展

Epstein-Barr病毒(EBV)感染是异基因造血干细胞移植(allo-HSCT)后的常见并发症,因其可能导致致死性并发症——移植后淋巴增殖性疾病(PTLD)而倍受关注.利妥昔单抗的应用,可以改善PTLD的预后,但EBV相关性中枢神经系统(CNS)-PTLD较为罕见,且目前尚无公认的有效治疗方案,因此其预后较其他部位PTLD差.现就EBV相关性CNS广PTLD的诊治进展进行综述如下,旨在探讨对该病合适的治疗手段.     

Epstein-Barr viral load measurement as a marker of EBV-related disease.

Epstein-Barr virus (EBV) is associated with a wide variety of benign and neoplastic diseases. EBV viral load assays that may prove useful in rapid assessment of disease status are now available. The two most common approaches to viral load measurement are quantitative, competitive PCR, and real-time PCR. Laboratory studies have shown that these assays are sensitive and specific for measuring EBV DNA in blood samples. Clinical investigations suggest a role for viral load measurement in predicting and monitoring EBV-associated tumors, including nasopharyngeal carcinoma, post-transplant lymphoproliferative disorder, Hodgkin's disease, and AIDS-related lymphoma. These new laboratory tools show promise in improving clinical management of affected patients.     

T cell-based therapies for EBV-associated malignancies

Epstein-Barr virus (EBV) is associated with a number of human malignancies. The cells of these tumours express a range of EBV latent cycle gene products that have the potential to be exploited as targets for T cell-mediated immunological therapies. Considerable progress has been made in developing adoptive T cell transfer for EBV-associated post-transplant lymphoproliferative disease (PTLD) and clinical experience clearly demonstrates that EBV-specific T cell responses can be used to treat this EBV-associated malignancy. Adoptive T cell therapies for other EBV-associated malignancies are less advanced, although encouraging data are starting to emerge. Adoptive T cell transfer, however, does require significant levels of specialist laboratory support. Large-scale treatment of patients in geographical areas with a high prevalence of EBV-associated malignancy is likely to require the development of therapeutic vaccination strategies, a number of which are in development at present. Although it remains to be seen whether long-lasting sterilising immunity to EBV could be achieved, an alternative vaccine-based approach would be to develop a prophylactic vaccine to protect against primary EBV infection.     

Infection of human endothelial cells with Epstein-Barr virus

Interleukin-6 (IL-6) promotes growth and tumorigenicity of Epstein-Barr virus (EBV)-immortalized B cells, and is abnormally elevated in the serum of solid organ transplant recipients who develop EBV-positive posttransplant lymphoproliferative disease (PTLD), but not in control transplant recipients. Endothelial cells derived from PTLD lesions were found to secrete spontaneously high levels of IL-6 in vitro for up to 4 mo. We examined possible mechanisms for sustained IL-6 production by endothelial cells. Here, we show that EBV can infect endothelial cells in vitro. After 3-4 wk incubation with lethally irradiated EBV- positive, but not EBV-negative cell lines, a proportion of human umbilical cord-derived endothelial cells (HUVECs) expressed in situ the EBV-encoded small RNAs (EBER). Southern blot analysis after polymerase chain reaction showed EBV DNA in HUVEC that had been incubated with lethally irradiated EBV-positive cells, but not in the controls. Exposure of HUVECs to lethally irradiated EBV-positive but not EBV- negative cell lines induced IL-6 production that was sustained for up to 120 d of culture. These studies identify endothelial cells as targets for EBV infection and raise the possibility that this infection may be important in the life cycle and pathology of EBV.     

Transforming activity and antigenicity of an Epstein-Barr-like virus from lymphoblastoid cell lines of baboons with lymphoid disease.

Two lymphoblastoid cell lines were established from baboons with lymphoid disease. Cells of these lines were positive for complement and Fc receptors but lacked sheep cell receptors, theraby indicating B-cell origin. The cells contained antigens which cross-reacted with Epstein-Barr virus (EBV), viral capsid antigen (VCA), early antigen (EA) and membrane antigen (MA). Both lines released virus with in vitro transforming activity for lymphocytes of several primate species including humans. Cells of the original lines and transformed cells showed no staining for EB nuclear antigen (EBNA). The virus was neutralized by anti-MA positive baboon and human sera. Baboon virus and EBV had different but overlapping in vitro host-cell ranges.     

Limitations of polymerase chain reaction testing for diagnosing acute Epstein-Barr virus infections

Clinicians use molecular tests to detect Herpesviridae from blood without fully appreciating limitations of testing. Studies are needed to enhance our understanding of the impact of Herpesviridae latency on molecular testing. We retrospectively performed quantitative Epstein-Barr virus (EBV) on sera from patients between the ages of 1 and 30 who demonstrated serologic evidence of acute EBV (n = 50) or remote EBV (n = 50) infection. Epstein-Barr virus DNA was detected in 70% of acutely infected and 4% of remotely infected patients. Sera from acutely infected patients had higher EBV copy number than convalescent sera. Our results suggest that serology should be performed as the initial diagnostic test for acute EBV. The role for polymerase chain reaction in immunocompromised patients with impaired antibody responses or as a 2nd-line diagnostic test when serologic results are equivocal deserves further study.     

Clinical reliability of IgG, IgA, and IgM antibodies in detecting Epstein-Barr virus at different stages of infection with a commercial nonrecombinant polyantigenic ELISA

We studied the diagnostic reliability of a modification of the Enzygnost EBV test (Behringwerke, Germany) for the detection of IgG, IgA, and IgM antibodies (Abs) in the diagnosis of Epstein-Barr virus (EBV) disease. One hundred and twenty-three serum samples were studied: 14 asymptomatic subjects without EBV infection, 48 patients with primary infection, 46 subjects with past EBV infection (11 patients with other acute infections), 8 patients without EBV infection but with other viral infection, and 7 patients with probable acute clonal stimulation of B lymphocytes caused by different microorganisms. Enzygnost EBV is based on an ELISA test with a pool of viral antigens. In our series the reliability of IgM for the diagnosis of recent primary EBV infection was: sensitivity 100%, specificity 95%, positive predictive value 90.5%, and negative predictive value 100%. The IgG detection with Enzygnost was: sensitivity 98%, specificity 100%, positive predictive value 100%, and negative predictive value 91.7%. Only two subjects had positive IgA. The Enzygnost test is an efficient method for the diagnosis of EBV infection although a few IgM false positives can occur.     

涎腺良、恶性淋巴上皮病变的临床病理特征及其与EB病毒的关系

目的 了解涎腺良、恶性淋巴上皮病变的临床病理特征及其与EB病毒的关系。方法 采用光镜、免疫组化染色、原位杂交、聚合酶链反应(PCR)，对14例涎腺恶性淋巴上皮病变(MLEL)和5例涎腺良性淋巴上皮病变(BLEL)进行观察，同时复习临床相关资料，并进行随访。结果 涎腺MLEL好发于腮腺、颌下腺，以女性为主，平均年龄47岁。组织学：大量增生的淋巴组织中见成簇或条索状分布的肿瘤细胞，瘤细胞界限不清，呈合体状，胞质嗜酸或淡染，核呈空泡状，核仁大而明显，核分裂象多见。免疫组化：瘤细胞表达CK；EBV阳性检出率92．9％，BLEL仅1例阳性。原位杂交：4例MLEL检出EBVEBERs；2例BLEL均为阴性。PCR：4例MLEL检出EBV／IR3，1例BLEL未检出。结论 涎腺MLEL是一类较少见、好发于腮腺、女性多见的未分化癌，组织学类似于鼻咽部的淋巴上皮癌。肿瘤的发生与EBV感染密切相关。     

Posttransplantation lymphoproliferative disease in children: otolaryngologic manifestations and management

BACKGROUND: Posttransplantation lymphoproliferative disease (PTLD) is associated with Epstein-Barr virus (EBV) infection after solid organ and bone marrow transplantation. METHODS: We did a retrospective analysis of cases with a diagnosis of PTLD at Children's Hospital of Wisconsin. RESULTS: Ten patients were identified. Seven of 10 cases (70%) were associated with bone marrow transplantation and 3 with solid organ transplantation. Three patients (30%) died of PTLD. The average time to development of PTLD after transplantation was 120 days. CONCLUSIONS: Otolaryngologic symptoms and findings are often the first manifestations of PTLD. Associated findings in this series included tonsillar necrosis, tonsillitis, airway obstruction, lymphadenitis, sinusitis, and otitis media. Diagnosis generally requires pathologic evaluation of tonsillar or adenoid tissue. Surgical intervention may also be important for relief of airway obstruction when present. Prompt recognition, diagnosis, and intervention with reduction in immunosuppression and antiviral therapy are essential to reduce the mortality of PTLD.     

Immediate Early and Early Lytic Cycle Proteins Are Frequent Targets of the Epstein-Barr Virus–induced Cytotoxic T Cell Response

Epstein-Barr virus (EBV), a human γ-herpesvirus, can establish both nonproductive (latent) and productive (lytic) infections. Although the CD8⁺ cytotoxic T lymphocyte (CTL) response to latently infected cells is well characterized, very little is known about T cell controls over lytic infection; this imbalance in our understanding belies the importance of virus-replicative lesions in several aspects of EBV disease pathogenesis. The present work shows that the primary CD8⁺ CTL response to EBV in infectious mononucleosis patients contains multiple lytic antigen-specific reactivities at levels at least as high as those seen against latent antigens; similar reactivities are also detectable in CTL memory. Clonal analysis revealed individual responses to the two immediate early proteins BZLF1 and BRLF1, and to three (BMLF1, BMRF1, and BALF2) of the six early proteins tested. In several cases, the peptide epitope and HLA-restricting determinant recognized by these CTLs has been defined, one unusual feature being the number of responses restricted through HLA-C alleles. The work strongly suggests that EBVreplicative lesions are subject to direct CTL control in vivo and that immediate early and early proteins are frequently the immunodominant targets. This contrasts with findings in α- and β-herpesvirus systems (herpes simplex, cytomegalovirus) where viral interference with the antigen-processing pathway during lytic infection renders immediate early and early proteins much less immunogenic. The unique capacity of γ-herpesvirus to amplify the viral load in vivo through a latent growth-transforming infection may have rendered these agents less dependent upon viral replication as a means of successfully colonizing their hosts.     

SURFACE MARKERS ON HUMAN B AND T LYMPHOCYTES : II. PRESENCE OF EPSTEIN-BARR VIRUS RECEPTORS ON B LYMPHOCYTES

Human peripheral lymphocytes were investigated for receptors binding Epstein-Barr virus (EBV) because of the regular association of this virus with infectious mononucleosis and Burkitt's lymphoma. This was done by a cytoadherence technique where virus-producing cells, displaying fresh viral determinants in their cytoplasmatic membrane, were mixed with lymphocytes. Unfractionated lymphocytes were found to adhere to these cells in contrast to column-purified T lymphocytes. The specificity of the binding was confirmed by blocking experiments that showed that sera containing high titers of antibodies directed against the virus could partially inhibit the adherence in contrast to low-titer sera. It is concluded that B lymphocytes, in contrast to T lymphocytes, have receptors for EBV. In a second line of experiments it was found that established human lymphoblastoid lines that carry the EBV genome had receptors characteristic for B lymphocytes and did not form T-lymphocyte rosettes. In contrast, a line of known T-lymphocyte origin that did not carry the EBV genome had receptors characteristic for T lymphocytes. EBV-transformed simian lymphoblastoid lines had surface markers indicating a B-lymphocyte origin in contrast to HVS-transformed simian lines that lacked surface immunoglobulin but carried receptors for sheep red blood cells.     

Dysregulated Epstein-Barr virus infection in the multiple sclerosis brain

Epstein-Barr virus (EBV), a ubiquitous B-lymphotropic herpesvirus, has been associated with multiple sclerosis (MS), an inflammatory disease of the central nervous system (CNS), but direct proof of its involvement in the disease is still missing. To test the idea that MS might result from perturbed EBV infection in the CNS, we investigated expression of EBV markers in postmortem brain tissue from MS cases with different clinical courses. Contrary to previous studies, we found evidence of EBV infection in a substantial proportion of brain-infiltrating B cells and plasma cells in nearly 100% of the MS cases examined (21 of 22), but not in other inflammatory neurological diseases. Ectopic B cell follicles forming in the cerebral meninges of some cases with secondary progressive MS were identified as major sites of EBV persistence. Expression of viral latent proteins was regularly observed in MS brains, whereas viral reactivation appeared restricted to ectopic B cell follicles and acute lesions. Activation of CD8⁺ T cells with signs of cytotoxicity toward plasma cells was also noted at sites of major accumulations of EBV-infected cells. Whether homing of EBV-infected B cells to the CNS is a primary event in MS development or the consequence of a still unknown disease-related process, we interpret these findings as evidence that EBV persistence and reactivation in the CNS play an important role in MS immunopathology.