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Laboratoire de Virologie Moléculaire et Structurale, EA , Université Joseph Fourier
Laboratoire de Virologie Médicale and Service de Maladies Infectieuses, Centre Hospitalier Universitaire, Grenoble
bioMérieux, Lyon, France
Epstein-Barr virus (EBV) DNA loads in peripheral blood mononuclear cells (PBMCs), plasma, and saliva, as well as infectivity of the virus in saliva, were evaluated in 20 patients for 6 months after the onset of infectious mononucleosis (IM). All patients displayed sustained high EBV DNA loads in the saliva, associated with a persistent infectivity of saliva at day 180. EBV DNA load in PBMCs decreased significantly from day 0 to day 180 (in spite of a viral rebound between day 30 and day 90 in 90% of the patients), and EBV DNA rapidly disappeared from plasma. These data show that patients with IM remain highly infectious during convalescence.
Infectious mononucleosis (IM) related to Epstein-Barr virus (EBV) primary infection is a self-limited lymphoproliferative disorder characterized by an expansion of EBV-infected B lymphocytes in lymphoid tissues and blood, associated with lytic replication of the virus in the oropharynx. B cell proliferation and viral replication are controlled by a vigorous CD8+ cytotoxic T cell immune response that is responsible for most of the clinical symptoms of the disease [13]. The primary infection is followed by a lifelong persistence of the virus, without viral production, in resting memory B lymphocytes, associated with recurrent release of infectious particles in the oral cavity; this recurrent release is the source of infection of EBV-naive individuals and is responsible for the periodic reinfection of B lymphocytes in the oral lymphoid tissue [3]. Close monitoring of virological data during the period between primary infection and long-term EBV carriage has not been common [47]. In the present study, blood and saliva from 20 patients with IM were prospectively collected until 6 months after the onset of symptoms, to determine the kinetics of EBV DNA load in blood and saliva and the long-term infectivity of saliva.
Subjects, materials, and methods.
We enrolled 20 previously healthy individuals with serologically confirmed IM (7 men and 13 women; mean age, 20 years; age range, 1834 years). Ten healthy EBV carriers (carrier status determined by the presence of IgG antibodies against EBV viral capsid antigen and EBNA and the absence of IgM against VCA) acted as control individuals in the study (4 men and 6 women; mean age, 23 years; age range, 1830 years). Informed consent was obtained from all patients and control individuals, and the study was approved by the Grenoble medical ethics committee (Comité Consultatif pour la Protection des Personnes en Recherche Biomédicale). Patients and control individuals were enrolled between September 2001 and April 2003.
EDTA peripheral blood and saliva (12 mL) samples were collected from patients with IM at the date of first consultation (day 0 [D0]), D3, D7, D15, D30, D60, D90, and D180. Similar samples were obtained from healthy EBV carriers. Peripheral blood mononuclear cells (PBMCs) and the plasma fraction were separated by use of a Ficoll-Hypaque gradient. All of the samples were frozen at -80°C until use. Measurement of EBV DNA load in saliva and blood samples was performed as described elsewhere [8], by LightCycler real-time quantitative polymerase chain reaction (PCR) with an amplification in the thymidine kinase gene, hybridization probe technology, and an external standard (serial dilutions of EBV DNA extracted from Namalwa cells). By use of this method, we were able to detect 5 copies of the EBV genome/reaction (linearity, 5500,000 copies/reaction).
To assess the infectivity of saliva, the saliva samples collected at D0 and D180 from each patient were simultaneously thawed, serially diluted, and titrated on the same fresh cord-blood lymphocytes, by use of the lymphocyte transformation assay without cyclosporin A. Each saliva dilution was tested in 8 replicates, and cord-blood lymphocytes were cultivated at 37°C (5% CO2) in RPMI 1640 medium supplemented with 20% fetal calf serum and were observed for EBV-induced foci of lymphoblastoid growth for 8 weeks. Titers are expressed in transformation units (TU) corresponding to the dilution of saliva that was sufficient to give a 50% incidence of cord-blood cell transformation by use of the Reed and Muench formula [9]. Virus released from the B95-8 cell line was used as positive control.
Statistical analyses were performed with the Statview J software package (version 4.02; Abacus Concepts). The EBV DNA loads in patients with IM and control individuals were compared using the unpaired t test, and the EBV DNA loads in patients with IM measured on different study days were compared with one another by use of the paired t test.
Results.
One hundred thirty-four PBMC, plasma, and saliva samples were collected from 20 patients with IM (P1P20). The average delay between the onset of IM symptoms and inclusion in the study (D0) was 2 days (range, 04 days). A short hospitalization was required for 8 patients because of more-severe disease, but only 1 of them received oral corticosteroid treatment, for 3 days. No antiviral treatment was used during the follow-up period. Eighty PBMC, plasma, and saliva samples were obtained from the control individuals.
High EBV DNA loads were observed in the saliva (figure 1A) from all patients with IM at D0 and D180 (median, 6.53 and 5.83 log copies/mL, respectively). Sixteen patients maintained a high EBV DNA load during the follow-up period, whereas the other 4 patients showed a decrease that ranged from 1 to 3 log copies/mL. This persistent high EBV DNA load in saliva during the 6 months after the beginning of IM contrasted with the low EBV DNA loads in saliva that were observed in the 10 healthy EBV carriers: 8 subjects had 24 episodes of detectable EBV DNA load (range, 1.74.9 log copies/mL), whereas the other 2 control subjects had no EBV DNA in their saliva during the follow-up period.
Sixteen patients with IM showed a positive EBV DNA load in PBMCs at every point during the follow-up period (figure 1B), whereas 4 patients showed a single episode of undetectable EBV DNA during the first month. The EBV DNA load observed in PBMCs of patients with IM at D0 ranged from 1.6 to 3.8 log copies/g DNA. A significant decrease was observed between D0 and D30 (P < .0001); the EBV DNA load observed at D30 in patients with IM was not significantly different from that observed in healthy EBV carriers (P = .19). However, an increase in EBV DNA load in PBMCs at D60 and/or D90 was observed in 18 patients (P < .001). Among these, 4 patients had tonsillitis and lymphadenopathy between D60 and D90. Whatever the fluctuations between D30 and D90, the EBV DNA load was significantly lower at D180 than at D90 in all patients (P = .02). Nine healthy control individuals showed a low fluctuating EBV DNA load in PBMCs, ranging from 0.9 to 1.8 log copies/g DNA. The tenth control individual showed no detectable EBV DNA in PBMCs during the entire follow-up period.
In plasma samples from all but 1 patient with IM, EBV DNA was detected at D0 (range, 1.43.3 log copies/mL) and decreased at D3; in all patients, EBV DNA was undetectable at D15 (figure 1C). EBV DNA loads in plasma but not in PBMCs or saliva tended to be significantly higher in hospitalized patients than in ambulatory patients at D0 (P = .05) and D3 (P = .03). The plasma specimens obtained from control individuals were always found to be negative.
As shown in table 1, all saliva samples tested were infective at D0 and D180. A trend toward a decrease in the infectivity titers was observed between D0 and D180 ( log range, 0.3 to 1.2 TU). The highest infectivity titers were observed in a single patient (P13), whereas the lowest infectivity titers were also observed in a single patient (P8), the one who harbored the highest EBV DNA load in saliva at D0. The infectivity titers were not statistically different between hospitalized and nonhospitalized patients.
Discussion.
To our knowledge, no longitudinal study has closely monitored EBV load in PBMCs, plasma, and saliva or the infectivity of saliva during the first 6 months after onset of IM. The data presented here highlight 2 points: (1) the persistence of high infectious EBV DNA loads in saliva during the follow-up period; and (2) a global decrease in EBV DNA loads in blood between D0 and D180, although a transient viral rebound occurred in PBMCs between D30 and D90, sometimes in association with clinical symptoms suggestive of IM recurrence.
In the present study, all patients with IM had EBV DNA in saliva at every point during the follow-up period, and these DNA loads were significantly higher than those in the healthy control individuals. At 6 months after onset of IM, 75% of the patients had EBV DNA loads that were still >5 log copies/mL, and only 20% showed a significant decrease in EBV DNA load between D0 and D180. We had the opportunity to assess the EBV DNA load in saliva from 5 patients with IM 1 year after enrollment. Two patients still had EBV DNA loads >5 log copies/mL, whereas the other 3 harbored loads similar to those in the healthy control individuals (data not shown). Similarly, saliva infectivity showed only a weak decrease between D0 and D180. In this transformation assay, the same cord-blood lymphocytes were used for the 2 samples from each patient, to avoid the variability in transformation titer described by Wilson et al. when different cord-blood samples are used to determine a virus titer [10]. Because of the small number of patients and the interpatient variability of this test, we were not able to correlate infectivity titers with EBV DNA load in saliva. Nevertheless, these results are in agreement with those of Yao et al., who demonstrated persistent infectivity of the saliva from 6 patients followed over 89 months after onset of IM [11]. Thus, the results of the present study suggest that EBV transmission could be more frequent through the saliva of an asymptomatic individual recently infected with EBV, compared with a long-term EBV carrier.
We demonstrated that the previously described higher EBV DNA load in PBMCs from patients with IM, compared with that control individuals, was no longer significant at D30 [4, 12]. We also showedfor the first time, to our knowledgea viral rebound in PBMCs by D60 or D90 in 90% of patients, associated with "recurrent IM symptoms" in 20% of the patients. Some early reports of "clinical recurrence of IM" have already been published, although without virological data [13, 14]. Although a superinfection with a different EBV strain could have been responsible for the IM symptom relapse [6], cloning and sequencing of the 3 end of the LMP-1 gene that distinguishes EBV strains did not show the appearance of new strains in 3 of the 4 patients at the time of their IM recurrence (data not shown). The progressive expansion of B lymphocytes expressing latent epitopes in the convalescent phase could also explain the increase in EBV DNA load observed in PBMCs. In this case, the development of CD8+ T cell responses against B lymphocytes expressing latent epitopes [2] might account for the clinical recurrence and the subsequent decrease in the EBV DNA load. Finally, the possibility that another viral or bacterial infection might be responsible for the clinical symptoms and for the reactivation of EBV has to be considered.
The follow-up assessment of EBV DNA load in plasma confirms previous reports of a transient presence of EBV DNA during the acute phase of IM and the possibility that plasma EBV DNA load is correlated with the severity of the disease as well as with the intensity of cellular immune reactions [1, 7, 8, 12]. The origin of EBV DNA in plasma during the acute phase of IM is not well understood. Ryan et al. [15] recently demonstrated that treating the EBV-positive plasma specimens by DNaseI did not abrogate PCR detection of EBV DNA, suggesting the presence of encapsidated DNA during IM, whereas naked EBV DNA was more frequently present in EBV-associated diseases in immunosuppressed patients. Furthermore, genotyping experiments showed that encapsidated EBV DNA in plasma during IM could arise from sources other than PBMCs [6]. These data suggested the presence of virus particles released in plasma during IM. Nevertheless, the clinical value of monitoring EBV DNA load in plasma during IM remains to be established.
Finally, the differences between EBV DNA loads in saliva and blood reported here, as well as the differences demonstrated by Nadal et al. between EBV DNA loads in sera and tonsils in healthy EBV carriers, support the view that mechanisms of EBV replication and specific anti-EBV immune responses in blood and in the oral cavity may be different ([16] and A. B. Rickinson, personal communication). The persistent high EBV DNA load in saliva suggests that patients remain highly infectious during IM convalescence, with different EBV DNA load patterns in saliva and blood. Immunovirological studies could be designed, for blood and saliva, to aid in understanding the different patterns of EBV DNA load in the 2 compartments and in exploring the difference between benign and severe IM.
Acknowledgments
We thank the general practitioners of the Centre Inter-Universitaire de la Médecine Préventive, Université Joseph Fourier, and Annick Bosseray from Département de Médecine Interne, Centre Hospitalier Universitaire, Grenoble, for providing the patients with IM; Marie-Annick Fernandes, for assistance in collecting samples; Marie-Josette Bourgeat and Geneviève Gui, for technical assistance; and Linda Northrup, for reviewing the English of this manuscript.
References
1. Williams H, Macsween K, McAulay K, et al. Analysis of immune activation and clinical events in acute infectious mononucleosis. J Infect Dis 2004; 190:6371. First citation in article
2. Hislop AD, Annels NE, Gudgeon NH, Leese AM, Rickinson AB. Epitope-specific evolution of human CD8+ T cell responses from primary to persistent phases of Epstein-Barr virus infection. J Exp Med 2002; 195:893905. First citation in article
3. Cohen JI. Epstein-Barr virus infection. N Engl J Med 2000; 343:48192. First citation in article
4. Maeda A, Wakiguchi H, Yokoyama W, Hisakawa H, Tomoda T, Kurashige T. Persistently high Epstein-Barr virus (EBV) loads in peripheral blood lymphocytes from patients with chronic active EBV infection. J Infect Dis 1999; 179:10125. First citation in article
5. Berger C, Day P, Meier G, Zingg W, Bossart W, Nadal D. Dynamics of Epstein-Barr virus DNA levels in serum during EBV-associated disease. J Med Virol 2001; 64:50512. First citation in article
6. Sitki-Green DL, Edwards RH, Covington MM, Raab-Traub N. Biology of Epstein-Barr virus during infectious mononucleosis. J Infect Dis 2004; 189:48392. First citation in article
7. Yamamoto M, Kimura H, Hironaka T, et al. Detection and quantification of virus DNA in plasma of patients with Epstein-Barr virus-associated diseases. J Clin Microbiol 1995; 33:17658. First citation in article
8. Brengel-Pesce K, Morand P, Schmuck A, et al. Routine use of real-time quantitative PCR for laboratory diagnosis of Epstein-Barr virus infections. J Med Virol 2002; 66:3609. First citation in article
9. Reed LJ, Muench H. A simple method for estimating fifty percent end points. Am J Hyg 1932; 27:4937. First citation in article
10. Wilson AD, Morgan AJ. Primary immune responses by cord blood CD4+ T cells and NK cells inhibit Epstein-Barr virus B-cell transformation in vitro. J Virol 2002; 76:507181. First citation in article
11. Yao QY, Ogan P, Rowe M, Wood M, Rickinson AB. Epstein-Barr virus-infected B cells persist in the circulation of acyclovir-treated virus carriers. Int J Cancer 1989; 43:6771. First citation in article
12. Fafi-Kremer S, Brengel-Pesce K, Bargues G, et al. Assessment of automated DNA extraction coupled with real-time PCR for measuring Epstein-Barr virus load in whole blood, peripheral mononuclear cells and plasma. J Clin Virol 2004; 30:15764. First citation in article
13. Chang RS, Maddock R. Recurrence of infectious mononucleosis. Lancet 1980; 1:704. First citation in article
14. Rose KD. Recurrence of infectious mononucleosis. JAMA 1972; 221:195. First citation in article
15. Ryan JL, Fan H, Swinnen LJ, et al. Epstein-Barr virus (EBV) DNA in plasma is not encapsidated in patients with EBV-related malignancies. Diagn Mol Pathol 2004; 13:618. First citation in article
16. Nadal D, Blasius M, Niggli FK, Meier G, Berger C. Epstein-Barr virus (EBV) DNA levels in palatine tonsils and autologous. J Med Virol 2002; 67:548. First citation in article