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Departments of Laboratory Medicine and Pathology and Pediatrics, University of Minnesota, Fairview Clinical Virology Laboratory, Departments of Medicine and Microbiology, Boynton Health Services
Department of Experimental and Clinical Pharmacology, University of Minnesota, Minneapolis
Background.
Characterizing virus-host interactions during self-limited infectious mononucleosis could explain how Epstein-Barr virus (EBV) replication is normally controlled and provide insight into why certain immunocompromised patients fail to contain it.
Methods.
University students had an average of 7 clinical and virologic evaluations during acute infectious mononucleosis. EBV was quantified in 697 samples of oral wash fluid, whole blood, peripheral blood mononuclear cells (PBMCs), and plasma by a real-time (TaqMan) polymerase chain reaction (qEBV) assay developed in our laboratory.
Results.
Twenty of 25 subjects had serologically confirmed primary EBV infection. EBV was cleared from whole blood by a first-order process with a median half-life of 3 days, and its quantity was associated with severity of illness (r2 = 0.82). Oral shedding persisted at a median of 1 × 104 copies/mL for 32 weeks and was unrelated to severity of illness. Subjects with nonprimary EBV infection shed virus intermittently, and median quantities for all samples became undetectable within 4 weeks.
Conclusions.
Using a novel qEBV assay, we demonstrated that young adults with primary EBV infection rapidly cleared virus from blood but not from the oropharynx. High oral concentrations of EBV in asymptomatic persons who have resumed normal activities support the concept that infectious mononucleosis is most likely acquired by kissing.
Primary Epstein-Barr virus (EBV) infection in adolescents and young adults often presents as acute infectious mononucleosis [1, 2]. From 1999 to 2003, the Boynton Health Service of the University of Minnesota cared for an average of 301 students with acute infectious mononucleosis annually (International Classification of Disease Revision 9, code 075). This large, on-campus patient population provided an ideal opportunity for us to study the viral dynamics of EBV infection in the human host by use of a real-time fluorogenic 5-nuclease (TaqMan) polymerase chain reaction (PCR) (qEBV) assay that we developed for monitoring EBV infection and disease. We reasoned that characterizing the viral-host interactions during self-limited acute infectious mononucleosis could enhance our understanding of how EBV replication is normally controlled. Such background information might provide insight into why certain immunocompromised patients fail to contain EBV infection and consequently develop severe or fatal disease.
SUBJECTS, MATERIALS, AND METHODS
Clinical study design.
University of Minnesota students who were 18 years old, had a clinical diagnosis of infectious mononucleosis, and presented within 10 days after the onset of symptoms were eligible to participate. The diagnosis was made by a student health service medical care provider and was usually based on the classic triad of fever, sore throat, and cervical lymphadenopathy. Students were excluded if they had had a previous acute illness that was consistent with infectious mononucleosis or if they were immunocompromised because of an underlying disease and/or receipt of immunosuppressive therapy. EBV infections were categorized on enrollment as primary, subacute, or previous according to the profile of antibodies against EBV viral capsid antigen (VCA) and Epstein-Barr nuclear antigen 1 (EBNA-1). Patients classified as having primary EBV infection were found to be positive for VCA IgM antibody and negative for EBNA-1 IgG antibody and either were found to be negative for VCA IgG antibody or had a lower quantity of VCA IgG antibody than VCA IgM antibody. Patients classified as having subacute EBV infection (due to persistence, reactivation, or reinfection) were found to be positive for all 3 antibodies. Patients classified as having previous EBV infection were found to be negative for VCA IgM antibody but positive for both VCA IgG antibody and EBNA-1 IgG antibody. All volunteers were encouraged to complete the study, which continued until 20 subjects with serologically confirmed primary EBV infection had enrolled. The subjects were seen at the Boynton Health Service or the Virology Research Clinic for clinical evaluations and sample collection. At each visit, a limited history was taken and a physical assessment was performed. The severity of illness was graded on a scale of 0 to 6, as defined in table 1. Clinic visits during the first academic year of the study were made twice weekly for 3 weeks, and a seventh visit was made 68 weeks after enrollment. A final visit 1 year after the onset of illness was requested from participants who returned to campus the following fall. During the second academic year of the study, subjects were scheduled for 7 visits: weekly for 4 weeks, at 6 weeks, at 12 weeks, and at 2442 weeks. The end point for the duration of events was the median time between the last day the event was present and the first day it was reported to be absent. This study was approved by the University of Minnesota Committee on the Use of Human Subjects in Research and the Boynton Health Service Research Committee. Informed consent was obtained from each subject before enrollment.
Collection and processing of oral wash fluid and blood.
At every visit, oral wash fluid and venous blood were obtained for use in the qEBV assay. On enrollment and at all visits after the first 3 weeks of illness, serum samples were tested for EBV antibodies. For oral wash fluid samples, subjects were instructed to rinse their mouths with 2030 mL of RPMI 1640 for 30 s and then expectorate into a collection cup. The oral wash fluid was centrifuged at 1000 g for 10 min, to remove bacteria and detritus. The solid matter remaining after the supernatant had been removed was resuspended in 1 mL of RPMI 1640 containing 10% fetal bovine serum, penicillin, and gentamicin. This solution was divided into 0.5-mL aliquots, and 2 dry cell pellets were made by centrifuging the samples at 14,000 g for 1.5 min. The excess fluid was removed with a Pasteur pipette. Peripheral blood mononuclear cells (PBMCs) were separated from EDTA-anticoagulated whole blood by Histopaque (Sigma-Aldrich) density-gradient centrifugation and were counted in a hemocytometer (American Optical). DNA was extracted from the oral wash fluidderived cell pellets, 2 × 1065 × 106 PBMCs, and 200 L each of whole blood, plasma, and the oral wash fluidderived supernatant by use of the QIAamp DNA Blood Mini Kit (Qiagen). DNA extractions were stored at -20°C until a complete set of samples from each patient could be assayed simultaneously. Aliquots of unextracted samples were stored at -80°C for future studies. The oral wash fluidderived cell pellet, the oral wash fluidderived supernatant, and whole blood samples were tested during both academic years of the study. PBMC and plasma samples were tested during only the first academic year of the study.
EBV antibody tests.
EBV antibody assays (VCA IgM, VCA IgG, and EBNA-1 IgG antibodies) were performed on serum samples by use of commercially available EIA kits and a MAGO Plus Automated EIA Processor (Diamedix). Results were expressed as the index value, which was the absorbance of the patient's sample divided by the mean absorbance of 3 replicate dilutions of a weakly positive control supplied by the manufacturer. Results of the VCA IgM, VCA IgG, and EBNA-1 IgG antibody assays were classified according to their index value as negative (<0.90), equivocal (0.901.09), or positive (1.10).
qEBV assay.
The amplicon was a 71-bp portion of the EBNA-1 gene. The primers and probe were designed with the assistance of Primer Express software (version 1.0; PE Applied Biosystems): 5-GACTGTGTGCAGCTTTGACGAT-3 (forward), 5-CGGCAGCCCCTTCCA-3 (reverse), and 5-(FAM)-TAGATTTGCCTCCCTGGTTTCCACCTATG-(TAMRA)-3 (probe). The 25-L solution used in the qEBV assay contained 5 L of patient DNA, 12.5 L of ABI TaqMan Universal PCR Master Mix (Applied Biosystems), 300 nmol/L of the forward and reverse primers, and 400 nmol/L of the probe. PCR was performed in an ABI Prism 7700 oligonucleotide sequence detector. The PCR conditions were 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Serial 10-fold dilutions, from 1 × 101 to 1 × 1011 copies, of the amplicon produced as a plasmid in Escherichia coli with the TOPO TA cloning kit (Invitrogen) were used to establish the standard curve. Controls included 2 dilutions of Namalwa cells (ATCC CRL1422), which contain 2 integrated copies of EBV per cell [3]; blood from patients known to have had EBV viremia previously (the diagnosis was confirmed by sending aliquots of the same sample to a reference laboratory at Eastern Virginia Medical School, Norfolk, Virginia); oral wash fluid and blood samples from an EBV-seronegative volunteer; and reactions without template. Quantitative EBV data were expressed as viral copies per milliliter for the oral wash fluidderived cell pellet, the oral wash fluidderived supernatant, whole blood, and plasma and as viral copies per 1 × 106 PBMCs. Preliminary experiments indicated that the reliable limit of detection of the assay (coefficient of variation, <30%) was 10 viral copies/reaction.
Statistics.
To calculate viral elimination rates, the log-linear portion of each individual's viral copy number versus the time curve was identified by visual inspection. Linear regression was performed on the log-transformed viral load versus day of illness data to obtain the rate constant (K) using Kaleidagraph data analysis software (version 3.6; Synergy Software). The half-life of the viral decay was computed as ln(2)/K. The half-life of the severity of illness scores was calculated similarly. The relationships between the severity of illness score and qEBV data for the oral wash fluidderived cell pellet and whole blood were also examined by regression analysis.
Because the severity of illness score is an ordered categorical variable rather than a continuous scale, we pooled data by low severity of illness scores (02) and high severity of illness scores (36) and compared the log-transformed qEBV data between these 2 groups using an unpaired t test (GraphPad InStat version 3; GraphPad Software). The test was performed separately for qEBV data from the oral wash fluidderived cell pellet and whole blood.
RESULTS
Demographics of the study population.
Twenty-five subjects (18 women and 7 men) participated from 5 December 2002 to 17 December 2004. The median age of the study subjects was 20.3 years (mean, 20.6 years [range, 1826 years]). Subjects enrolled a median of 5 days (mean, 5.1 days [range, 29 days]) after the onset of illness. Primary EBV infection was confirmed by EBV antibody testing in 20 subjects. Two subjects who were experiencing persistence, reactivation, or reinfection with EBV were categorized as having subacute EBV infection. Three subjects had evidence of previous EBV infection. The median number of study visits per subject was 7 (mean, 6.4 study visits/subject). The median follow-up period was 49 days (mean, 144 days). The subjects provided a total of 697 samples for the qEBV assay (median, 30 samples/subject; mean, 28 samples/subject).
Clinical findings and severity of illness.
The 20 subjects with primary EBV infection had a median severity of illness score of 4 (mean, 3.6 [range, 06]) on enrollment (figure 1). The 5 subjects with nonprimary EBV infection also had an initial median severity of illness score of 4 (mean, 3.6 [range, 25]). Subjects with primary EBV infection could not easily be distinguished from those with nonprimary EBV infection on the basis of symptoms and physical findings (table 2). Pharyngitis was the most common physical finding, and tiredness was the most frequent complaint. Abdominal pain and nausea were more common and the duration of sore throat and headache was longer in subjects with nonprimary EBV infection. This was reflected by the trajectories of the severity of illness scores, which showed that the rate of recovery for subjects with nonprimary EBV infection was slower than that for subjects with primary EBV infection (figure 1). There was no sex difference in the severity of illness scores or the qEBV data from any of the compartments tested (data not shown).
Antibody patterns during EBV infection.
The median EBV antibody EIA indices for the 25 subjects are displayed in table 3. Eighteen of the 20 subjects with primary EBV infection had positive VCA IgM antibody indices on enrollment. One subject had a negative VCA IgM antibody index (0.16) when she presented on the seventh day of illness, but her VCA IgM antibody index became positive (5.88) a week later. Another subject had an equivocal VCA IgM antibody index (1.09) when she enrolled on the seventh day of illness. Six weeks later, she had a positive VCA IgM antibody index (1.34) but still had negative VCA IgG and EBNA-1 IgG antibody indices. Because of her slow humoral immune response to primary EBV infection, she was retested 11 months after the onset of illness, at which time she had an EBV antibody profile that was typical of someone with a previous EBV infection (VCA IgM antibody index, 0.81; VCA IgG antibody index, 4.00; and EBNA-1 IgG antibody index, 3.95). VCA IgG and EBNA-1 IgG antibodies became detectable relatively late during the course of infection, and VCA IgM antibodies tended to linger. At 7 weeks after the onset of illness, 12 (71%) of 17 subjects with primary EBV infection still had positive VCA IgM antibody indices, 13 (76%) had positive VCA IgG antibody indices, and none had positive EBNA-1 IgG antibody indices. At 12 weeks after the onset of illness, 6 (71%) of 7 subjects with primary EBV infection had positive VCA IgM and VCA IgG antibody indices, but only 3 (43%) of these 7 subjects had positive EBNA-1 IgG antibody indices. At 32 weeks after the onset of illness, all 5 subjects with primary EBV infection who were tested had positive VCA IgG and EBNA-1 IgG antibody indices, but 2 of them still had positive VCA IgM antibody indices.
In 1 of 2 subjects with subacute EBV infection, the VCA IgM antibody index decreased from 2.02 to 0.92, and the EBNA-1 IgG antibody index increased from 3.24 to 5.55. In the other subject with subacute EBV infection and in all 3 subjects with previous EBV infection, no appreciable change was observed in any of the 3 EBV antibody indices measured.
The median half-life of viral elimination from whole blood in 19 subjects was 3.0 days (mean, 3.5 days [range, 1.86.6 days]). For 1 subject, only 1 whole blood qEBV assay was performed, and, thus, a half-life could not be estimated. In 4 subjects, the quantity of EBV in whole blood increased during the first 2 weeks, and the quantities measured before the maximum quantity was reached were not included in the regression analysis. The median half-life of the improvement in the severity of illness score was 5.9 days (mean, 5.0 days [range, 1.38.7 days]).
The elimination of EBV from the oropharynx was very different from the elimination of EBV from whole blood and PBMCs. Virus persisted at an elevated level for 32 weeks in both the oral wash fluidderived cell pellet and the oral wash fluidderived supernatant. However, individual patterns demonstrated interesting trends. Quantitative viral data were obtained between 90 and 419 days after the onset of illness in 12 subjects. At the time of the last measurement during this longer follow-up period, 5 subjects continued to have EBV in their oral wash fluid, whereas EBV was not detectable in the oral wash fluid from the other 7 subjects. The viral load in the oral wash fluidderived cell pellet decreased in 14 of the 20 subjects at some point during the first 3 weeks of illness but later rebounded. The second peak in the viral load was actually higher than the first peak in 6 of these 14 subjects. No second peak was observed in 4 of these 20 subjects, and too few samples were obtained from the remaining 2 subjects to enable us to discern a pattern.
Finally, the median quantities of EBV in plasma were always below the level of detection. Only 12 (14%) of 84 plasma samples from subjects with primary EBV infection contained virus, and the quantity of virus in the positive samples was relatively low (mean, 3700 copies/mL [range, 10009900 copies/mL]).
The median (range) log10 copies of EBV in whole blood in the group with low severity of illness scores was 3.55 log10 copies/mL (2.704.65 log10 copies/mL). In the group with high severity of illness scores, the median (range) was 4.52 log10 copies/mL (3.045.56 log10 copies/mL). This was statistically significant (P < .0001). Interestingly, the difference in the median (range) log10 copy number in the oral wash fluidderived cell pellet between the groups was also significant, but it was in the opposite direction: high severity of illness scores were associated with lower quantities of EBV in the oral wash fluidderived cell pellet. In the group with low severity of illness scores, the median (range) was 4.88 log10 copies/mL (2.906.67 log10 copies/mL), compared with 4.51 log10 copies/mL (2.305.51 log10 copies/mL) in the group with high severity of illness scores (P = .03). The log-transformed EBV DNA values were found to have equal variances (P > .05) between the groups with high and low severity of illness scores and also passed the Kolmogorov-Smirnov test, and this showed that data were sampled from Gaussian distributions (P > .10) in both the oral wash fluidderived cell pellet and whole blood.
Detection and quantitation of EBV in nonprimary versus primary infection.
The 5 subjects who had previous EBV infection shed virus intermittently. None of the 5 subjects had EBV detected in >1 whole blood sample, and only 2 subjects had EBV detected in oral wash fluid on consecutive occasions. The quantity of EBV detected in the samples from various sites was also lower than that found in subjects with primary EBV infection, with plasma being a notable exception (table 4). Plasma viremia was uncommon in both primary and nonprimary EBV infection: overall, only 15 (13%) of 116 plasma samples were found to have EBV.
DISCUSSION
A major finding of this study was that the quantity of EBV in the blood was related to the clinical severity of primary EBV infection but that the quantity of EBV in the oral wash fluid was not. Although our data are insufficient to prove cause and effect, they support the concept that the quantity of EBV in whole blood samples is a surrogate marker for the severity of primary EBV infection in young adults. The rate of viral elimination from whole blood was similar to the rate of improvement in the severity of illness score (figures 1 and 2), which suggests that monitoring the change in viremia might predict the duration of symptoms in primary EBV infection.
Oropharyngeal excretion of EBV has been well documented in persons with both acute and previous EBV infection [47]. Oral shedding has also been shown to persist for many months after the onset of infectious mononucleosis [5, 6, 8, 9]. However, the present study provides the important additional observation that the quantity of virus in oral secretions was not related to the severity of illness and may, in fact, have an inverse relationship. Oral shedding was continuous rather than intermittent, and both the cell pellet and the supernatant derived from oral wash fluid contained a median of 1 × 104 copies/mL during 32 weeks of observation. If the quantity of detectable virus correlates with infectivity, patients may be just as infectious during convalescence as when they are acutely ill. In support of this possibility, saliva collected 180 days after the onset of infectious mononucleosis has been shown to be infectious on the basis of an in vitro cord-blood lymphocyte transformation assay [9]. Because 18 of our 20 subjects with primary EBV infection became asymptomatic (severity of illness score, 0) within 4 weeks after the onset of illness, the majority of young adults appear to be asymptomatic for the majority of time they are shedding EBV in the oropharynx. Finding large quantities of EBV in the oral wash fluid from young adults who most likely had resumed normal social activities is completely consistent with Hoagland's observation "that infectious mononucleosis is usually transmitted as a result of intimate oral contact which permits a transfer of saliva" ([10], p. 271). In other words, infectious mononucleosis is aptly named "the kissing disease."
Where in the oral compartment is EBV produced There is evidence to support the salivary glands [11], oropharyngeal epithelial cells [12, 13], and, more recently, B cells in the oropharynx [14, 15]. Both cell-free and cell-associated EBV were present in our subjects. For approximately one-third of the oral wash fluid samples, more EBV was found in the derived supernatant than in the derived cell pellet, suggesting that certain cells in the oral compartment are capable of producing and releasing a large quantity of extracellular viral material.
Our data suggest that EBV replication is not as tightly controlled by EBV-specific immunity in the oropharynx as it is in the blood. This may be because cell entry is enhanced via a receptor switch when EBV alternatively infects B cells and epithelial cells [16]. By improving its efficiency of infection and switching the cell type producing it, EBV circumvents immune recognition in the oropharynx to provide a relatively long-lasting pool of infectious virions.
Our study contributes to the understanding of EBV infection in the immunocompromised host by providing quantitative data on the level and duration of viremia and oral shedding in immunocompetent young adults throughout the course of primary EBV infection. Because the peak viral loads were 5.6 log10 copies/mL in whole blood and 6.7 log10 copies/mL in oral wash fluid from our subjects, it is clear that having a single high viral load does not signal the host's loss of control of EBV infection. Tissue damage and the evolution of malignancy most likely depend on the duration of viral replication or the total exposure to virus over time (the area under the viral concentrationtime curve). Perhaps this could be monitored in the oral compartment as a first, noninvasive step, which immunocompromised children would find to be especially agreeable. A prospective study that correlates the quantity of EBV in the blood and oral compartments with EBV infection might improve existing laboratory testing strategies for high-risk patients. Plasma viremia was present for only a short time, if at all, in our subjects with self-limited infection. Hence, plasma viremia may prove to be a good indicator of the host's loss of control of EBV infection [17, 18].
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