Introduction
The World Health Organization's International Agency for Research on Cancer has designated the Epstein-Barr virus (EBV) as a group 1 carcinogenic agent, based on sufficient evidence for its causal role in the pathogenesis of several lymphoid and epithelial malignancies [1]. Paradoxically, EBV seroprevalence worldwide is over 90% and infection in the general population is rarely pathogenic. After primary infection, the virus establishes life-long and in most cases asymptomatic persistence, whereby it circulates in resting memory B cells in a quiescent state [2]. The number of circulating EBV-infected cells remains low and remarkably stable throughout life at approximately 0.5-50 EBV-positive cells per million B cells [3,4]. This reflects the tightly controlled balance between EBV-driven B-cell proliferation and the host's immune response. The latter is characterized by IgG reactivity to the EBNA1 and viral capsid antigen (VCA) p18 (BFRF3) proteins and by high levels of T cells reactive to latent and lytic cycle EBV antigens [5,6]. Disturbance of this balance, for example by T-cell immunosuppression or in immunological disorders may lead to EBV-DNA load elevations in the blood. For example, we demonstrated elevated circulating EBV-DNA loads in lung transplant recipients with posttransplant lymphoproliferative disease, which were directly related to increased numbers of circulating EBV-infected B cells [7]. Similarly, elevated circulating EBV-DNA loads are detectable in patients with EBV-DNA-positive AIDS-related non-Hodgkin's lymphoma (ARNHL) but not in EBV-DNA-negative (yet EBV-seropositive) cases [8-11]. Elevated EBV-DNA loads are also observed in approximately 20% of asymptomatic HIV carriers, in the absence of EBV-linked morbidity [12-17]. Absolute EBV-DNA load detection in the general HIV-infected population thus has poor diagnostic value for ARNHL risk stratification.
The origin and source of the elevated EBV-DNA loads remain undetermined, and thus far it is unclear how EBV persists at an increased level in the circulation of HIV carriers without causing morbidity. Therefore, an objective of this study on EBV-HIV co-infection was to investigate whether EBV-DNA loads are persistently elevated in HIV carriers approximately 10 years after the introduction of HAART. We aimed to determine the source of the circulating EBV DNA, and define the EBV-infected cell. Furthermore, we studied EBV gene expression in the peripheral blood of HIV-seropositive individuals, to gain insight into the determinants of a potentially altered EBV latency phenotype in the circulation. In addition, EBV-RNA profiling has potential diagnostic value as it may discriminate 'normal' EBV homeostasis in the blood, characterized by relative transcriptional quiescence of EBV [2,18] from direct EBV-encoded oncogene-driven B-cell proliferation, as seen in ARNHL tissue biopsies [19]. Finally, we investigated whether anti-EBV serological responses are altered in the HIV population, as a reflection of disturbed or aberrant viral persistence.
Methods
Patients and clinical specimens
Random asymptomatic HIV carriers (N = 197) visiting the Slotervaart Hospital (Amsterdam, the Netherlands) for routine check-up during 2004-2006 were enrolled in the study. Whole blood (approximately 10 ml) was collected for routine diagnostic testing for plasma HIV-RNA load and CD4 T-cell counts in these individuals. Blood that was left over was used for EBV research purposes described below. Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll and stored in liquid nitrogen until use. Plasma was stored at -20°C. Approval for this study was obtained from the Medical Ethical Board of Slotervaart Hhospital (approval no. U/1859/0402). Only clinical specimens from adults who gave written informed consent were included in the study.
Peripheral blood mononuclear cell fractionation
B lymphocytes were isolated from PBMC using Dynabeads M-450 CD19 (Dynal Biotech, Smestad, Norway) according to the manufacturer's protocol. From the remaining cells (designated as 'non-B-cell fraction'), the CD3 cell fraction (designated as 'T cells') was isolated by Dynabeads M-450 CD3. On average 5 × 106 PBMC were used per donor. To determine the purity of isolated fractions, standard flow cytometry was performed on a FacsCalibur Instrument (BD Biosciences, Franklin Lakes, New Jersey, USA).
Epstein-Barr virus DNA and RNA isolation
DNA was isolated from clinical samples by silica-based extraction as described previously [20]. In addition, for increased concentration the DNA was input in polymerase chain reaction (PCR) for plasma specimens using the Qiamp DNA blood mini kit (Qiagen, Hilden, Germany) was used according to the manufacturer's instructions. RNA was isolated using RNA-Bee (Tel-Test; Friendswood, Texas, USA).
Epstein-Barr virus DNA load quantification
EBV-DNA copy numbers in clinical samples were determined using standardized quantitative LightCycler-based real-time PCR assays, targeting conserved 213 base pair (bp) or 99 bp regions of the viral EBNA1 gene, as described in detail recently [21,22].
Epstein-Barr virus RNA amplification
Nucleic acid sequence-based amplification (NASBA) was used to amplify EBV-encoded EBNA1, LMP1 and LMP2 genes and the low-copy cellular U1A snRNP housekeeping gene, as described previously [23]. Transcripts encoding the VCA-p18 (BFRF3) were amplified by NASBA using primers VCA-p18NAS1T7 (5′-aattctaatacgactcactataggggaggctgctaatagatgaagaaaca-3′) and VCA-p18NAS2P2 (5′-ccaacgcgccatagacaagaggca-3′). NASBA is a sensitive, isothermal RNA amplification technique that enables specific RNA amplification in a high DNA background, regardless of RNA splice patterns [24]. NASBA reagents were obtained from BioMerieux (NucliSens basic kit; BioMerieux, Boxtel, the Netherlands) and used according to their recommendations. Transcripts from the BamHI-A region of the EBV genome (BART) were amplified by reverse transcriptase (RT)-PCR using the A3/A4 primer set [25]. The RNA equivalent of 2.5*10E6 cells was used as input. Analytical sensitivities of NASBA and RT-PCR assays were previously described by us [23,26].
Anti-Epstein-Barr virus IgG and IgA determination
IgG and IgA antibody reactivity to VCA-p18 (BFRF3) and EBNA1 (BKRF1) were determined in duplicate by semiquantitative enzyme-linked immunosorbent assay (ELISA), clinically validated by us previously for Hodgkin's lymphoma and nasopharyngeal carcinoma patients [27-29]. These ELISA are based on synthetic peptides combining immunodominant epitopes derived from EBV-encoded EBNA1 and VCA-p18, identified previously using PepScan technology [6,29] (J.M. Middeldorp, unpublished data). Positive controls for IgG and IgA ELISA consisted of sera from healthy EBV-seropositive donors and nasopharyngeal carcinoma patients, respectively. Negative controls consisted of healthy EBV-seronegative donors. ELISA cut-off values were defined as the mean of three negative control sera plus 2 standard deviations. ELISA optical density (OD) values were normalized by dividing the mean of duplicate measurements for the clinical sample by the cut-off value.
Results
Persistently elevated circulating Epstein-Barr virus DNA loads in a fixed subset of asymptomatic HIV carriers
Whole blood of 197 asymptomatic HIV-seropositive individuals was sampled in the period 2004-2006, and 123 (62.4%) were positive for circulating EBV DNA as determined by EBV LightCycler PCR. Thirty-eight volunteers (19.3% of the total cohort) exhibited EBV-DNA loads above the previously determined cut-off value of 2000 EBV-DNA copies/ml blood, the upper limit in healthy HIV-seronegative EBV carriers [7].
EBV-DNA loads were unrelated to peripheral blood CD4 T-cell counts (Spearman's rho -0.231; P = 0.10) or plasma HIV-RNA loads (Spearman's rho 0.15; P = 0.915), confirming our previous study [12]. The HIV-RNA load in plasma correlated negatively with CD4 T-cell counts (Spearman's rho -0.416; P = 0.002). As shown in Fig. 1, the EBV-DNA load did not differ significantly between HIV carriers with CD4 T-cell counts below or above 250 cells/μl (Mann-Whitney; P = 0.64), whereas the HIV-RNA load in plasma was higher in HIV carriers with CD4 T-cell counts less than 250 cells/μl (Mann-Whitney; P = 0.03).
To investigate whether circulating EBV-DNA loads were persistently elevated, we collected a follow-up sample from 16 HIV-infected individuals that exhibited elevated EBV-DNA loads in 1999 [12] and that were still in the routine 3-monthly diagnostic follow-up of the Slotervaart Hospital, Amsterdam. Thirteen out of 16 HIV carriers (82%) still had elevated EBV-DNA loads in blood after approximately 5 years. As controls 23 randomly chosen HIV carriers who had undetectable EBV DNA in the blood in 1999 were included. Of these, only two (8%) had detectable EBV-DNA loads in 2004, at relatively low levels (2000-2800 copies/ml), whereas 21 patients (91%) remained negative for EBV DNA in the blood (see Table 1). This indicates that elevation of the circulating EBV-DNA load is a persistent phenomenon in a fixed subset of HIV-infected individuals, unrelated to EBV-associated morbidity or HIV activity.
Circulating Epstein-Barr virus DNA in HIV carriers is cell associated
To investigate whether the elevated EBV-DNA loads in the circulation of HIV-seropositive donors were associated with the cellular or the plasma compartment, we analysed 24 plasma samples, which were prepared from the same blood donation on which whole blood EBV-DNA loads were determined. These plasma samples were obtained from 24 randomly chosen HIV-infected individuals with elevated EBV-DNA loads in the corresponding whole blood sample (mean 4386 copies/ml; range 410-23 873 copies/ml). Our previous small pilot study already showed that plasma samples are mostly EBV-DNA negative [12]. Therefore, to enable the detection of EBV DNA in plasma by the most sensitive method, we used the Qiamp DNA blood mini kit for DNA isolation, which enables concentration of the plasma samples by a factor of approximately 8. A recently developed quantitative EBNA1-based LightCycler PCR, targeting a 99 bp region of this gene, was used for quantitation. This enables the detection of fragmented EBV DNA (< 180 bp) derived from apoptosed cells, which for example predominates in the circulation of nasopharyngeal carcinoma patients [22,30].
Only one out of 24 plasma samples was positive for EBV DNA (300 copies/ml). Spiking of the negative samples with fixed amounts of EBNA1 DNA as described previously [31] gave positive PCR results in all cases, indicating the absence of PCR inhibitors in the plasma preparations. A positive control plasma sample, obtained from a patient with EBV-positive lymphoproliferative disease, gave a strong fluorescent signal in LightCycler PCR (10 760 copies/ml plasma), whereas plasma samples from healthy EBV-seropositive donors were negative. These data confirm that EBV DNA in the blood of HIV-infected patients is absent from the plasma and is therefore contained within the cellular compartment.
Epstein-Barr virus DNA in asymptomatic HIV carriers mainly circulates in B cells
As described above, the EBV DNA in the circulation of HIV-infected individuals is restricted to the cellular blood compartment. To define the EBV-infected cells, we used Dynabeads to isolate B cells and T cells from PBMC of HIV-seropositive donors from which sufficient clinical material was available. For each cell fraction, the EBV-DNA load was determined. As shown in Fig. 2, a clear enrichment of EBV DNA was seen in the purified B-cell fraction for all tested samples. The EBV-DNA load per millilitre of blood was not correlated with the percentage of total CD20-positive B cells in individual patients (Spearman's rho 0.09; P = 0.78), indicating that increased EBV-DNA loads are not simply caused by a higher number of circulating B cells.
Circulating Epstein-Barr virus-infected cells in HIV carriers have restricted Epstein-Barr virus transcription
To assess whether the cell-associated EBV genomes are transcriptionally active, NASBA and RT-PCR were used to amplify EBV RNA from PBMC. These analyses could be completed for 22 random HIV carriers who had elevated EBV-DNA loads and for whom sufficient PBMC were available (EBV-DNA load range 336-44 900 copies/ml blood). All PBMC specimens tested positive for the low copy human U1A snRNP housekeeping messenger RNA, indicating good RNA quality. For EBV transcriptional profiling, we focussed on the non-translated A3/A4 spliced transcript originating from the BART, the EBV latent mRNA encoding for EBNA1, LMP1 and LMP2 and a transcript representative of the lytic EBV replication cycle, encoding the VCA-p18 (BFRF3). BART were detectable in 15 out of 22 samples (68%), again confirming that the elevated EBV-DNA loads in the blood of HIV carriers are cell associated (Fig. 3). EBNA1 and LMP1 transcripts were never detectable in PBMC fractions. LMP2 transcripts were found in 11 out of 22 samples (50%), whereas eight of these were also positive for BART. The results for BART and LMP2 RNA analyses in relation to EBV-DNA loads are depicted in Fig. 4. Transcripts encoding VCA-p18 were not detectable in PBMC samples, despite positivity for U1A snRNP mRNA, BART RNA and elevated EBV-DNA loads in these specimens.
Asymptomatic HIV carriers have an aberrant anti-Epstein-Barr virus serological profile characterized by frequent IgA to Epstein-Barr virus VCA-p18
Using synthetic peptide-based ELISA we determined IgG and IgA reactivity to immunodominant epitopes derived from EBNA1 and VCA-p18. All asymptomatic HIV carriers tested (n = 54) were positive for IgG to VCA-p18 (normalized ELISA OD value range of 2.29-16.68; median 13.71), showing 100% EBV seropositivity in this Dutch HIV cohort, as could be expected. Anti-EBNA1 IgG was present in 50 out of 54 (94%) at a normalized ELISA OD range of 1.16-12.13 (median 3.95). HIV carriers with elevated circulating EBV-DNA loads (> 2000 copies/ml) had significantly elevated IgG reactivity to VCA-p18 compared with HIV carriers with EBV-DNA loads less than 2000 copies/ml (P < 0.0001). In contrast, anti-EBNA1 IgG levels were significantly lower in the high EBV-DNA load group (P = 0.003), confirming our previous study in HIV carriers [12]. At the quantitative level, EBV-DNA copies/ml blood significantly correlated with the anti-VCA-p18 IgG ELISA OD value (Spearman's rho 0.60; P < 0.0001) and inversely correlated with the anti-EBNA1 IgG ELISA OD value (Spearman's rho -0.44; P = 0.001). Individual IgG responses to VCA-p18 and EBNA1 were inversely correlated (Spearman's rho -0.438; P < 0.0001).
Remarkably, the majority of HIV carriers showed IgA reactivity to VCA-p18, which was found in 37 out of 54 donors (69%), with a normalized ELISA OD range of 1.07-8.84 (median 4.63) in the positive samples. EBNA1-specific IgA was found in 22 HIV carriers (40%) but at low levels (normalized ELISA OD range of positive samples was 1.01-2.65; median 1.28). At the qualitative level, HIV carriers with elevated circulating EBV-DNA loads showed a trend towards higher anti-VCA-p18 IgA levels (P = 0.09). VCA-p18-specific IgA levels and EBV-DNA copies/ml blood were correlated (Spearman's rho 0.32; P = 0.018). Serology results are shown in Fig. 5. Finally, normalized positive OD values for the VCA-p18 IgA ELISA were unrelated to those for VCA-p18 IgG (Spearman's rho 0.214; P = 0.12). Similar results were found for responses to EBNA1 (Spearman's rho 0.149; P = 0.51), indicating differential antigenic triggering of anti-EBV IgA and IgG in individual patients.
Discussion
We investigated the persistence and molecular characteristics of EBV infection in the circulation of asymptomatic HIV carriers and analysed a large cohort, representing 2% of the total registered Dutch HIV population (see 'Fact Sheet SOA en HIV' [in Dutch] at www.rivm.nl). Our results substantiate previous findings that approximately 20% of HIV carriers have elevations of EBV DNA in the blood [12-17]. These elevated EBV-DNA loads are persistent in a fixed subset of HIV carriers, without indications of any EBV-related morbidity or overt immune dysfunction. The increased EBV-DNA loads are B-cell associated and are unrelated to direct productive EBV infection in the circulation. Moreover, high EBV-DNA load carriers show aberrant humoral immune responses to EBV, characterized by significantly higher IgG and frequent IgA reactivity to VCA-p18. We found that IgA and IgG reactivity to this EBV capsid antigen are quantitatively unrelated. This points to differential antigenic triggering of B cells producing the two Ig subclasses. Although difficult to investigate in vivo, this phenomenon may be related to the site of antigen presentation (mucosal versus lymph nodes or epithelial versus lymphoid EBV-infected cells) or the source of VCA-p18 (virion-derived immune-complexed protein versus host cell-expressed protein).
Increased anti-VCA-p18 IgG levels in HIV carriers with high EBV-DNA loads suggest increased lytic EBV replication. This view is consistent with a study by Piriou et al. [16] showing that increased EBV-DNA loads after HIV seroconversion are paralleled by increased T-cell numbers reactive to early lytic EBV antigens. The site of lytic EBV replication in HIV carriers is probably not the peripheral blood because we now show that neither virions (e.g. cell-free viral DNA) nor EBV lytic cycle transcripts are detectable in plasma and PBMC, respectively. We hypothesize that lytic replication occurs in the lymphoid tissues of the oropharynx or their subepithelial linings. The oropharynx is the preferential homing site for EBV-infected B cells in healthy EBV-seropositive donors [32,33]. Our hypothesis is further supported by the fact that asymptomatic HIV carriers have increased EBV shedding in the saliva, paralleled by increased lytic viral replication in oropharyngeal epithelial cells [17,34-36]. In AIDS patients this is particularly evident when presenting with oral hairy leukoplakia, a benign epithelial lesion showing a unique combination of transforming and permissive EBV activity [37]. In HIV-negative EBV carriers, the amount of EBV shedded into the saliva is much lower, and lytic viral replication in oropharyngeal epithelial cells is a rare event [38], reflected in low/undetectable circulating EBV-DNA loads [2,5,7]. The oropharynx as a site for increased lytic EBV replication in HIV infection is further supported by our findings of frequent anti-VCA-p18 IgA positivity, indicating a mucosal source of the VCA-p18 antigen. IgA to EBV proteins is rarely seen in healthy EBV carriers [29,39] arguing for aberrant EBV persistence in the HIV population.
The trigger for EBV lytic replication in HIV carriers may be linked to EBV infection of epithelial cells or cross-linking of the B-cell receptor (BCR) on EBV-infected cells by antigen, leading to plasma cell differentiation [33,40,41]. Ig genes of circulating EBV-infected B cells are isotype switched and hypermutated, and thus display the molecular hallmarks of antigen specificity [42]. Possibly, the encounter with (opportunistic) antigens in lymphoid tissue during B-cell homing may temporarily switch on EBV lytic replication. Interaction of EBV-infected B cells with CD4 T cells may also initiate lytic viral replication via the CD40-CD40 ligand (gp39) signalling route [43]. Interestingly, HIV carriers have decreased circulating memory B-cell numbers but generally higher Ig levels, suggestive of increased plasma cell formation [44].
The observed EBV-RNA profile suggests that EBV in the circulation of HIV-infected individuals persists at the transcriptional level in a similar way to healthy EBV-seropositive donors [45-48], which is in an inactive, non-immunogenic reservoir. Only the number of EBV-infected cells in the blood of HIV carriers is higher as determined from the increased cell-associated EBV-DNA loads attributed to the B-cell compartment. The presence of EBV latent RNA in PBMC confirms that circulating EBV DNA is cell associated. The highly restricted EBV transcriptional phenotype in the blood of HIV carriers is similar to that found in HIV-negative, EBV-seropositive donors [46,47].
Circulating BART were detectable in the majority of HIV carriers with elevated EBV-DNA loads. BART are transcribed in all EBV-associated diseases [49] and also in the circulation of healthy EBV-seropositive individuals [47,48], but their function remains undefined. We previously showed that the predicted BARF0 open reading frame within the BART is not translated in vivo, suggesting a direct role for the RNA itself [25]. BART might function as antisense transcripts and prevent translation of the complementary BALF, which encode (immunogenic) proteins of the lytic cycle. In addition, predicted introns in BART contain microRNA, with a postulated role in regulating cellular or viral gene expression [50].
Like healthy EBV carriers, the HIV-infected donors in this study often had detectable circulating LMP2 mRNA. LMP2 may act as a surrogate BCR and provide constitutive signals required for survival [51]. Work in transgenic mice showed that LMP2 can be compatible with BCR signalling and can bypass normal B-cell developmental checkpoints [51]. In addition, LMP2 segregates Syk and Lyn protein tyrosine kinases and blocks BCR translocation into lipid membrane rafts [51-53], thereby preventing signal transduction after BCR cross-linking by antigen and thus maintaining a resting phenotype. As BCR activation may induce the EBV lytic cycle [40,41], LMP2 may be important for maintaining EBV latency and preventing cytotoxic elimination of the EBV-infected cell by T cells reactive to highly immunogenic early lytic cycle antigens. The absence of (cell-free) EBV DNA in plasma, that is the absence of virions and negativity for VCA-p18 transcripts indicated the absence of systemic lytic EBV replication in the presented HIV population.
LMP1 mRNA was never detected in the circulation of HIV carriers. This proves that the increased number of EBV-carrying cells is not caused by their direct proliferation in the circulation [2]. In transplant recipients a similar viral transcriptional phenotype was observed, indicating that proliferating B-blasts, expressing the EBV growth programme do not accumulate in the blood [2,54]. We could, however, detect LMP1 mRNA in transplant recipients with lymphoma, accompanied by extremely high EBV-DNA loads and blastoid EBV-positive B cells in the circulation, indicating that this EBV transcription phenotype is only tolerated at severe immunosuppression [7].
EBNA1 mRNA was absent from all PBMC samples, in parallel with previous findings in both healthy donors and immunosuppressed patients [46-48,54]. EBNA1 transcription is only seen when circulating EBV-carrying memory B cells divide, which is an infrequent event [18,55]. During cell division, EBNA1 protein is required to ensure confidential EBV replication and the equal division of EBV genomes over the daughter cells [18]. The absence of EBNA1 mRNA again proves that EBV-infected cells do not actively replicate in the circulation of HIV carriers. This would imply that the increased numbers of EBV-infected cells are generated elsewhere in the body. We postulate two, not mutually exclusive mechanisms for this: (i) the direct increased proliferation of EBV-infected B cells in lymph nodes differentiating into memory B cells and accumulating in the blood; or (ii) increased lytic EBV replication and neo-infection of B cells in lymph nodes/epithelial cells, subsequently differentiating into memory B cells. Unfortunately, these hypotheses are difficult to test because ethical restrictions do not allow lymphoid tissues to be obtained from asymptomatic HIV carriers to perform EBV transcript analyses or immunohistochemical staining for defining EBV latency or lytic replication in situ.
The clinical consequences of persistently elevated circulating EBV-DNA loads in a fixed subset of HIV carriers are unclear. It is tempting to speculate that an increased EBV burden increases the risk of EBV-driven B-cell derailment and eventually of lymphoma. On the other hand, these EBV-carrying cells may not provide a pathogenic threat to the host because they circulate in a resting state and do not proliferate. Testing these hypotheses would require a study of EBV gene expression in peripheral lymphoid (tonsillar) tissue, preferably at lymphadenopathy, and immunophenotyping of the EBV-infected cells, combined with large epidemiological follow-up studies on the incidence of ARNHL. This should be related to the evolving characteristics of HIV infection, anti-HIV treatment efficacy and the presence of opportunistic pathogens that may provide antigenic stimuli to EBV-carrying B cells.
Acknowledgements
The authors would like to thank the technicians of the Department of Molecular Biology, Slotervaart Hospital, Amsterdam for collecting clinical specimens.
Conflicts of interest: None.
References
1. International Agency for Research on Cancer. IARC monographs on the evaluation of carcinogenic risks to humans, vol. 70: Epstein-Barr virus and Kaposi's sarcoma herpesvirus/human herpesvirus 8. Lyon, France: IARC; 1997.
2. Thorley-Lawson DA. Epstein-Barr virus: exploiting the immune system. Nat Rev Immunol 2001; 1:75-82.
3. Joseph AM, Babcock GJ, Thorley-Lawson DA. EBV persistence involves strict selection of latently infected B cells. J Immunol 2000; 165:2975-2981.
4. Thorley-Lawson DA. EBV: schizophrenic but not dysfunctional. EBV Rep 1999; 6:158-163.
5. Young LS, Rickinson AB. Epstein-Barr virus: 40 years on. Nat Rev Cancer 2004; 4:757-768.
6. Van Grunsven WM, Spaan WJ, Middeldorp JM. Localization and diagnostic application of immunodominant domains of the BFRF3-encoded Epstein-Barr virus capsid protein. J Infect Dis 1994; 170:13-19.
7. Stevens SJC, Verschuuren EAM, Pronk I, Van der Bij W, Harmsen M, The TH, et al. Frequent monitoring of EBV DNA load in unfractionated whole blood is essential for early detection of PTLD in high risk patients. Blood 2001; 97:1165-1171.
8. Stevens SJC, Vervoort MBHJ, Van den Brule AJC, Meenhorst PL, Meijer CJLM, Middeldorp JM. Monitoring of Epstein-Barr virus DNA load in peripheral blood by quantitative competitive polymerase chain reaction. J Clin Microbiol 1999; 37:2852-2857.
9. Laroche C, Drouet EB, Brousset P, Pain C, Boibieux A, Biron F, et al. Measurement by the polymerase chain reaction of the Epstein-Barr virus load in infectious mononucleosis and AIDS-related non-Hodgkin's lymphomas. J Med Virol 1995; 46:66-74.
10. Fan H, Kim SC, Chima CO, Israel BF, Lawless KM, Eagan PA, et al. Epstein-Barr viral load as a marker of lymphoma in AIDS patients. J Med Virol 2005; 75:59-69.
11. Bonnet F, Jouvencel AC, Parrens M, Leon MJ, Cotto E, Garrigue I, et al. A longitudinal and prospective study of Epstein-Barr virus load in AIDS-related non-Hodgkin lymphoma. J Clin Virol 2006; 36:258-263.
12. Stevens SJC, Blank BSN, Smits PHM, Meenhorst PL, Middeldorp JM. High Epstein-Barr virus (EBV) DNA loads in HIV-infected patients: correlation with antiretroviral therapy quantitative EBV serology. AIDS 2002; 16:993-1001.
13. Van Baarle D, Hovenkamp E, Callan MF, Wolthers KC, Kostense S, Tan LC, et al. Dysfunctional Epstein-Barr virus (EBV)-specific CD8(+) T lymphocytes and increased EBV load in HIV-1 infected individuals progressing to AIDS-related non-Hodgkin lymphoma. Blood 2001; 98:146-155.
14. Dehee A, Asselot C, Piolot T, Jacomet C, Rozenbaum W, Vidaud M, et al. Quantification of Epstein-Barr virus load in peripheral blood of human immunodeficiency virus-infected patients using real-time PCR. J Med Virol 2001; 65:543-552.
15. Righetti E, Ballon G, Ometto L, Cattelan AM, Menin C, Zanchetta M, et al. Dynamics of Epstein-Barr virus in HIV-infected subjects on highly active antiretroviral therapy. AIDS 2002; 16:63-73.
16. Piriou ER, Van Dort K, Nanlohy NM, Miedema F, Van Oers MH, Van Baarle D. Altered EBV viral load setpoint after HIV seroconversion is in accordance with lack of predictive value of EBV load for the occurrence of AIDS-related non-Hodgkin lymphoma. J Immunol 2004; 172:6931-6937.
17. Ling PD, Vilchez RA, Keitel WA, Poston DG, Peng RS, White ZS, et al. Epstein-Barr virus DNA loads in adult human immunodeficiency virus type 1-infected patients receiving highly active antiretroviral therapy. Clin Infect Dis 2003; 37:1244-1249.
18. Hochberg D, Middeldorp JM, Catalina M, Sullivan JL, Luzuriaga K, Thorley-Lawson DA. Demonstration of the Burkitt's lymphoma Epstein-Barr virus phenotype in dividing latently infected memory cells in vivo. Proc Natl Acad Sci U S A 2004; 101:239-244.
19. Brink AA, Dukers DF, Van den Brule AJ, Oudejans JJ, Middeldorp JM, Meijer CJ, et al. Presence of Epstein-Barr virus latency type III at the single cell level in posttransplantation lymphoproliferative disorders and AIDS related lymphomas. J Clin Pathol 1997; 50:911-918.
20. Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, Van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990; 28:495-503.
21. Stevens SJC, Verkuijlen SAWM, Middeldorp JM. Quantitative detection of Epstein-Barr virus DNA in clinical specimens by rapid real-time PCR targeting a highly conserved region of EBNA-1. Meth Mol Biol 2005; 292:15-25.
22. Stevens SJC, Verkuijlen SAWM, Hariwiyanto B, Harijadi A, Fachiroh J, Paramita D, et al. Diagnostic value of measuring Epstein-Barr virus (EBV) DNA load and carcinoma-specific viral mRNA in relation to anti-EBV immunoglobulin A (IgA) and IgG antibody levels in blood of nasopharyngeal carcinoma patients from Indonesia. J Clin Microbiol 2005; 43:3066-3073.
23. Brink AATP, Vervoort MBHJ, Middeldorp JM, Meijer CJLM, Van den Brule AJC. Nucleic acid sequence-based amplification, a new method for analysis of spliced and unspliced Epstein-Barr virus latent transcripts, and its comparison with reverse transcriptase PCR. J Clin Microbiol 1998; 36:3164-3169.
24. Deiman B, Van Aarle P, Sillekens P. Characteristics applications of nucleic acid sequence-based amplification (NASBA). Mol Biotechnol 2000; 20:163-167.
25. Van Beek J, Brink AA, Vervoort MB, Van Zijp MJ, Meijer CJ, Van den Brule AJ, et al. In vivo transcription of the Epstein-Barr virus (EBV) BamHI-A region without associated in vivo BARF0 protein expression in multiple EBV-associated disorders. J Gen Virol 2003; 84:2647-2659.
26. Stevens SJC, Brink AATP, Middeldorp JM. Profiling of Epstein-Barr virus latent RNA expression in clinical specimens by gene-specific multiprimed cDNA synthesis and PCR. Methods Mol Biol 2005; 292:27-38.
27. Meij P, Vervoort MB, Bloemena E, Schouten TE, Schwartz C, Grufferman S, et al. Antibody responses to Epstein-Barr virus-encoded latent membrane protein-1 (LMP1) and expression of LMP1 in juvenile Hodgkin's disease. J Med Virol 2002; 68:370-377.
28. Karray H, Ayadi W, Fki L, Hammami A, Daoud J, Drira MM, et al. Comparison of three different serological techniques for primary diagnosis and monitoring of nasopharyngeal carcinoma in two age groups from Tunisia. J Med Virol 2005; 75:593-602.
29. Fachiroh J, Paramita DK, Hariwiyanto B, Harijadi A, Dahlia HL, Indrasari SR, et al. Single-assay combination of Epstein-Barr Virus (EBV) EBNA1- and viral capsid antigen-p18-derived synthetic peptides for measuring anti-EBV immunoglobulin G (IgG) and IgA antibody levels in sera from nasopharyngeal carcinoma patients: options for field screening. J Clin Microbiol 2006; 44:1459-1467.
30. Chan KC, Zhang J, Chan AT, Lei K, Leung SF, Chan LY, et al. Molecular characterization of circulating EBV DNA in the plasma of nasopharyngeal carcinoma lymphoma patients. Cancer Res 2003; 63:2028-2032.
31. Stevens SJC, Pronk I, Middeldorp JM. Toward standardization of Epstein-Barr virus DNA load monitoring: unfractionated whole blood as preferred clinical specimen. J Clin Microbiol 2001; 29:1211-1216.
32. Pegtel DM, Middeldorp J, Thorley-Lawson DA. Epstein-Barr virus infection in ex vivo tonsil epithelial cell cultures of asymptomatic carriers. J Virol 2004; 78:12613-12624.
33. Laichalk LL, Thorley-Lawson DA. Terminal differentiation into plasma cells initiates the replicative cycle of Epstein-Barr virus in vivo. J Virol 2005; 79:1296-1307.
34. Brengel-Pesce K, Morand P, Schmuck A, Bourgeat MJ, Buisson M, Bargues G, et al. Routine use use of real-time quantitative PCR for laboratory diagnosis of Epstein-Barr virus infections. J Med Virol 2002; 66:360-369.
35. Miller CS, Berger JR, Mootoor Y, Avdiushko SA, Zhu H, Kryscio RJ. High Prevalence of multiple human herpesviruses in saliva from human immunodeficiency virus-infected persons in the era of highly active antiretroviral therapy. J Clin Microbiol 2006; 44:2409-2415.
36. Walling DM, Flaitz CM, Nichols CM, Hudnall SD, Adler-Storthz K. Persistent productive Epstein-Barr virus replication in normal epithelial cells in vivo. J Infect Dis 2001; 184:1499-1507.
37. Webster-Cyriaque J, Middeldorp J, Raab-Traub N. Hairy leukoplakia: an unusual combination of transforming and permissive Epstein-Barr virus infections. J Virol 2000; 74:7610-7618.
38. Herrmann K, Frangou P, Middeldorp J, Niedobitek G. Epstein-Barr virus replication in tongue epithelial cells. J Gen Virol 2002; 83:2995-2998.
39. Ng WT, Yau TK, Yung RW, Sze WM, Tsang AH, Law AL, Lee AW. Screening for family members of patients with nasopharyngeal carcinoma. Int J Cancer 2005; 113:998-1001.
40. Takada K. Cross-linking of cell surface immunoglobulins induces Epstein-Barr virus in Burkitt lymphoma lines. Int J Cancer 1984; 33:27-32.
41. Daibata M, Humphreys RE, Takada K, Sairenji T. Activation of latent EBV via anti-IgG-triggered, second messenger pathways in the Burkitt's lymphoma cell line Akata. J Immunol 1990; 144:788-793.
42. Souza TA, Stollar BD, Sullivan JL, Luzuriaga K, Thorley-Lawson DA. Peripheral B cells latently infected with Epstein-Barr virus display molecular hallmarks of classical antigen-selected memory B cells. Proc Natl Acad Sci U S A 2005; 2:18093-18098.
43. Fu Z, Cannon MJ. Functional analysis of the CD4(+) T-cell response to Epstein-Barr virus: T-cell-mediated activation of resting B cells and induction of viral BZLF1 expression. J Virol 2000; 4:6675-6679.
44. De Milito A, Morch C, Sonnerborg A, Chiodi F. Loss of memory (CD27) B lymphocytes in HIV-1 infection. AIDS 2001; 15:957-964.
45. Takeuchi H, Kobayashi R, Hasegawa M, Hirai K. Detection of latent infection by Epstein-Barr virus in peripheral blood cells of healthy individuals and in nonneoplastic tonsillar tissue from patients by reverse transcription-polymerase chain reaction. J Virol Methods 1996; 58(1-2):81-89.
46. Tierney RJ, Steven N, Young LS, Rickinson AB. Epstein-Barr virus latency in blood mononuclear cells: analysis of viral gene transcription during primary infection and in the carrier state. J Virol 1994; 68:7374-7385.
47. Chen H, Smith P, Ambinder RF, Hayward SD. Expression of Epstein-Barr virus BamHI-A rightward transcripts in latently infected B cells from peripheral blood. Blood 1999; 93:3026-3032.
48. Yang J, Tao Q, Flinn IW, Murray PG, Post LE, Ma H, et al. Characterization of Epstein-Barr virus-infected B cells in patients with posttransplantation lymphoproliferative disease: disappearance after rituximab therapy does not predict clinical response. Blood 2000; 96:4055-4063.
49. Smith P. Epstein-Barr virus complementary strand transcripts (CSTs/BARTs) and cancer. Semin Cancer Biol 2001; 11:469-476.
50. Cai X, Schafer A, Lu S, Bilello JP, Desrosiers RC, Edwards R, et al. Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed. PLoS Pathogen 2006; 2:e23.
51. Ikeda M, Fukuda M, Longnecker R. Function of latent membrane protein 2A. In: Robertson ES, editor. Epstein-Barr virus. Norfolk, UK: Caister Press; 2005.
52. Dykstra ML, Longnecker R, Pierce SK. Epstein-Barr virus coopts lipid rafts to block the signaling and antigen transport functions of the BCR. Immunity 2001; 14:57-67.
53. Miller CL, Lee JH, Kieff E, Longnecker R. An integral membrane protein (LMP2) blocks reactivation of Epstein-Barr virus from latency following surface immunoglobulin crosslinking. Proc Natl Acad Sci U S A 1994; 91:772-776.
54. Babcock GJ, Decker LL, Freeman RB, Thorley-Lawson DA. Epstein-Barr virus-infected resting memory B cells, not proliferating lymphoblasts, accumulate in the peripheral blood of immunosuppressed patients. J Exp Med 1999; 190:567-576.
55. Schittek B, Rajewsky K. Maintenance of B-cell memory by long-lived cells generated from proliferating precursors. Nature 1990; 346:749-751.
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