Host factors associated with the kinetics of Epstein-Barr virus DNA load in patients with primary Epstein-Barr virus infection

Authors


Tetsushi Yoshikawa, Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi 4701192 Japan. Tel: +81 562 939251; fax: +81 562 952216; email: tetsushi@fujita-hu.ac.jp

ABSTRACT

The aims of this study were to elucidate the kinetics of Epstein-Barr virus (EBV) DNA load in serially collected peripheral blood mononuclear cells of patients with primary EBV infection, and to determine the correlated host factors. Blood samples were collected from 24 patients with primary EBV infection. EBV DNA copy numbers were measured using real-time polymerase chain reaction. Based on the kinetics of EBV DNA load, the 24 patients were divided into two groups: rapid regression and slow regression. Eighteen of the 24 patients (75%) were included in the slow regression and 6 (25%) in the rapid regression group. No statistically significant differences were observed between the two groups in clinical features and laboratory findings. However, acute phase (3 to 10 days after the onset of the illness) serum samples from six children in the slow regression and four in the rapid regression group revealed significantly higher serum interleukin (IL)-1β (P= 0.018), IL-12 (P= 0.009), tumor necrosis factor-α (P= 0.019), interferon-inducible protein 10, and monokine induced by interferon γ concentrations in the rapid regression than the slow regression group. On the other hand, sera from six children in the slow regression and four in the rapid regression group in the convalescent phase (14 to 21 days after the onset of the illness) showed no statistically significant differences between the two groups in these biomarker concentrations. Based on this, it was concluded that the kinetics of EBV DNA load can be divided to two different patterns after primary EBV infection, and immune response might be associated with viral clearance.

List of Abbreviations: 
CBA

cytometric bead array

EBV

Epstein-Barr virus

IFN

interferon

IL

interleukin

IM

infectious mononucleosis

IP

interferon inducible protein

MIG

monokine induced by interferon-γ

PBMCs

peripheral blood mononuclear cells

TNF

tumor necrosis factor

Epstein-Barr virus (EBV) is a gamma-herpesvirus that is ubiquitous in humans. Primary EBV infections are largely asymptomatic in infants; however, some children and young adults with primary viral infection present as infectious mononucleosis (IM). The typical clinical features of IM are fever, lymphadenopathy, tonsillopharyngitis, hepatosplenomegaly, and malaise. Since other pathogenic agents cause similar clinical features, a serological assay is required for confirmation of EBV-associated IM. After primary infection, EBV can remain latent throughout life and reactivate in immunocompromised patients such as transplant recipients, resulting in post-transplant lymphoproliferative disorder (1).

Real-time PCR is useful for the diagnosis of EBV-associated disease in transplant recipients (2–4). Therefore, measurement of EBV DNA load in peripheral blood using real-time PCR has become popular in the management of transplant recipients. Although several investigators have reported that this method is reliable for the diagnosis of primary EBV infection (5–8), the kinetics of EBV DNA load in the peripheral blood in such patients remains unclear. Therefore, in this study, we measured EBV DNA load in serially collected peripheral blood mononuclear cells (PBMCs) from patients with primary EBV infection to elucidate the kinetics of EBV DNA load. We also analyzed the correlations between the kinetics of EBV DNA and host clinical features, laboratory findings, and serum cytokine and chemokine concentrations.

MATERIALS AND METHODS

Patients, inclusion criteria and definition of groups

Twenty-four children (14 boys and 10 girls) with primary EBV infection were enrolled prospectively after obtaining informed consent from their patients and guardians. The median age of the subjects was 6 years (range 1–
16 years). This study was approved by the review board at our university. All patients had clinical symptoms suggestive of IM and were admitted to the pediatric departments of Fujita Health University Hospital or Toyokawa Municipal Hospital between October 2006 and September 2009. The diagnosis of IM was based on clinical findings and the following serological results: positive for IgG and/or IgM anti-EB viral capsid antigen antibodies and negative for IgG anti-EB nuclear antigen antibody. EDTA-treated peripheral blood (2 mL) and serum (1 mL) were serially collected from all patients.

The patients were separated into two groups based on real-time PCR assessment of EBV DNA load. The slow regression group was defined as having high copy numbers of viral DNA (>500 copies/μg DNA) in PBMCs 21 days after the onset of illness, whereas the rapid regression group was defined as having low copy numbers of viral DNA (<500 copies/μg DNA) 21 days after the onset of the illness.

Sample preparation and DNA extraction

PBMCs were isolated from EDTA-treated peripheral blood. DNA was extracted from the PBMCs using the QIAamp blood kit (Qiagen GebH, Hilden, Germany), and eluted in 50 μL of elution buffer, then stored at −30°C until analysis.

Real-time polymerase chain reaction

Real-time PCR reactions were performed using a TaqMan PCR kit (PE Applied Biosystems, Foster City, CA, USA). The PCR primers for this assay were selected in the BALF5 gene encoding the viral DNA polymerase (6). A standard curve was generated using the CT values obtained from serially diluted pGEM-BALF5. Copy numbers for the clinical samples were calculated automatically by Sequence Detector version 1.6 software. The detection limit of the real-time PCR was 10 copies/reaction.

Analysis of clinical features and laboratory findings

In order to elucidate host factors that might regulate the kinetics of EBV DNA load in PBMCs, clinical symptoms at the time of admission to the hospitals, such as fever (>38.0°C), lymphadenopathy, tonsillopharyngitis, skin rash, and hepatosplenomegaly, were retrospectively collected from the medical records. Additionally, duration of fever was assessed to determine disease severity. Other laboratory data that were collected from the medical records included the maximum values of aspartate aminotransferase and alanine aminotransferase concentrations, white blood cells counts, and ratio of atypical lymphocytes during the observation period.

Measurement of cytokine and chemokine concentrations

Serum cytokine and chemokine concentrations were measured in samples collected in the acute (between 3 and 10 days of the illness) and convalescent (between 14 and 21 days of the illness) phases of the disease. The nine cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IFN-γ, TNF-α) and five chemokines (IL-8, IP-10, monocyte chemotactic protein-1, MIG, regulated on activation normal T-cell expressed and secreted) were measured by the CBA system (BD Biosciences, San Jose, California, USA). Assay standards or patient sera (50 μL) were added to 50 μL of a cocktail of capture beads and detector antibodies, and the mixture incubated for 1.5 hr at room temperature in the dark. Excess unbound detector antibody was removed by washing, and 50 μL of reagent added before data acquisition. Two-color flow-cytometric analysis was performed (FACScan, Becton Dickinson, Franklin Lakes, NJ, USA). One thousand eight hundred events were acquired by following the protocol supplied and analysis was performed using CBA dedicated analysis software (CellQuest, Becton Dickinson). All samples for which the calculated cytokine concentration was below the given assay sensitivity were treated as undetectable.

Statistical analysis

Age and laboratory data were compared between the slow and rapid regression groups by Student's t-test. Sex and clinical features were examined by Fisher's exact test. Cytokine and chemokine concentrations were compared between the two groups using the Wilcoxon signed-rank test. Statistical analysis was performed with JMP software, version 7.

RESULTS

The kinetics of Epstein-Barr virus copy numbers in peripheral blood mononuclear cells

We divided the 24 patients into two groups based on the criteria defined above in the ‘Patients, inclusion criteria and definition of groups’ section of Patients and Methods. The kinetics of EBV DNA copy numbers in these two groups are shown in Figure 1. Eighteen of the twenty-four patients (75%) were included in the slow regression group (Fig. 1a), and 6 (25%) in the rapid regression group (Fig. 1b).

Figure 1.

Kinetics of EBV DNA load in PBMCs as assessed by real-time PCR. (a) Slow regression group; high numbers of copies of viral DNA (>500 copies/μg DNA) were detected in PBMC 21 days after the onset of the illness. (b) Rapid regression group; viral DNA had decreased quickly (<500 copies/μg DNA) by 21 days after the onset of illness. Vertical dotted lines indicate 21 days after the onset of the illness, and horizontal dotted lines indicate 500 copies of EBV DNA/μg DNA.

Clinical features and laboratory findings

We compared the clinical features and laboratory data between the two groups (Table 1). Although fever was the commonest clinical feature (22/24, 91.7%), lymphadenopathy (14/24, 58.3%), tonsillopharyngitis (13/24, 54.1%), skin rash (6/24, 25.0%), and hepatosplenomegaly (6/24, 25.0%) were also seen. However, we observed no statistically significant difference between the two groups in the frequency of the various clinical features assessed. Mean durations of fever in the slow and rapid regression groups were 8.4 and 8.8 days, respectively (P= 0.844). Additionally, we observed no statistically significant age and sex differences between the two groups.

Table 1.  Clinical features and laboratory findings were compared between slow and rapid regression groups
CategoriesEBV viral load in PBMCP value
Slow regression (n= 18)Rapid regression (n= 6)
  1. *The maximum value of each item during the observation period is shown.

  2. ALT, alanine aminotransferase; AST, aspartate aminotransferase; wbc, white blood cells.

Age (years)6.7 ± 4.86.0 ± 2.60.788
Sex (male/female)12/62/40.151
Fever16/186/61.000
Lymphadenopathy10/184/61.000
Tonsillopharyngitis10/183/61.000
Skin rash5/181/61.000
Hepatosplenomegaly3/181/61.000
Duration of fever (days)8.4 ± 4.38.8 ± 4.10.844
AST*(IU/L)197.1 ± 174.1432.5 ± 705.60.665
ALT*(IU/L)248.8 ± 211.2273.5 ± 295.10.868
WBCs*(/μL)15083.3 ± 6889.4 15183.3 ± 3829.1 0.841
Atypical lymphocytes* (%)15.9 ± 21.220.5 ± 18.20.525
EBV DNA load in PBMCs*(copies/μg)482616.1 ± 175957.5206109.1 ± 420865.60.665

Typical abnormal laboratory findings, such as increase in hepatic transaminase concentrations, leukocytosis, and increased numbers of atypical lymphocytes were observed. However, we demonstrated no statistically significant differences in laboratory findings between the two groups.

Measurement of cytokine and chemokine concentrations

Differences in cytokine concentrations between the two groups are shown in Table 2. Acute phase (3 to 10 days after the onset of illness) serum samples were available for measurement of the biomarkers in 10 children (six in the slow and four in the rapid regression group). There was no statistically significant difference in sampling time between the two groups (slow regression group; 6 ± 3.7 days, rapid regression group; 5.8 ± 3.1 days, P= 0.914). In this phase, the rapid regression group had significantly higher serum concentrations of IL-1β (P= 0.018), IL-12 (P= 0.009), TNF-α (P= 0.019), IP-10 (P= 0.042), and MIG (P= 0.019) than the slow regression group. On the other hand, in the convalescent phase (14 to 21 days after the onset of the illness) sera from 10 children (six in the slow and four in the rapid regression group) revealed no statistically significant differences between the two groups in any of the cytokines and chemokine concentrations. Again, there was no statistically significant difference in sampling time between the two groups (slow regression group; 16.3 ± 0.7 days, rapid regression group; 16.5 ± 0.9 days, P= 0.89).

Table 2.  Serum cytokine and chemokine concentrations in the slow and rapid regression groups
 Acute phase (Days 3–10) EBV viral load in PBMCsConvalescent phase (Days 14–21) EBV viral load in PBMCs
Slow regression (n= 6)Rapid regression (n= 4)P valueSlow regression (n= 6)Rapid regression (n= 4)P value
  1. *Concentrations of biomarkers are shown (median 25%–75%) in this table.

  2. Acute phase (3 to 10 days after the onset of the illness) serum samples were available for measurement of the biomarkers in 10 children (six in the slow and four in the rapid regression group).

  3. Ten convalescent phase (14 to 21 days after the onset of the illness) sera were available in 10 children (six in the slow and four in the rapid regression group). Bold indicates the cytokine or chemokine concentrations for which we observed statistically significant differences between the two groups.

  4. RANTES, regulated on activation normal T-cell expressed and secreted.

Cytokine (pg/mL)*
 IL-1β1.98 (0–4.6) 13.1 (11.4–16.4) 0.0185.4 (4.2–6.9)4.7 (4.6–5.5)0.915
 IL-20 (0–0)0 (0–5.2)  0.6480 (0–9.7)0 (0–0)  0.223
 IL-40 (0–0)0 (0–2.2)  0.8790 (0–0)  0 (0–0)  1.000
 IL-50 (0–0)0 (0–0)    0.4140 (0–4.5)0 (0–0)  0.224
 IL-6   9.7 (7.3–13.0)22.9 (14.1–29.8) 0.08712.2 (5.9–23.7)4.9 (4.4–9.9)0.394
 IL-10  11.6 (6.9–15.2)17.7 (13.9–143.3)0.286 7.6 (3.6–13.7)3.7 (3.6–3.8)0.394
 IL-121.4 (0–3.0)14.4 (13.1–15.6) 0.0095.1 (3.4–7.5)3.2 (3.1–3.3)0.109
 IFN-γ0 (0–0)0 (0–10.3) 0.4140 (0–0)  0 (0–0)  0.414
 TNF-α  5.9 (3.8–7.2)10.6 (1.0–12.8)  0.0197.8 (4.5–9.4)3.7 (3.5–5.6)0.522
Chemokine (pg/mL)*
 IL-8   40.0 (12.4–71.9)100.9 (45.3–1320.8)0.200 46.1 (16.7–77.3) 9.0 (7.2–83.8)0.394
 IP-10    1282.3 (436.4–2032.1) 3499.5 (2446.7–5744.8)0.042  1269.6 (314.5–2313.0)      201.3(108.1–404.7)0.201
 MCP-1  26.9 (3.4–87.9)251.4 (121.7–416.0)0.05523.8 (3.8–72.5)3.8 (3.7–4.4)0.394
 MIG    1737.4 (366.6–3223.8)  8449.8 (7211.3–41281.8)0.019  1177.2 (133.8–3305.9)  77.9 (44.8–184.8)0.201
 RANTES     15165.4 (7679.6–20609.0)  19493.8 (16736.0–19854.9)0.670   8621.8 (6558.8–13246.8)   7040.6 (6677.0–7202.5)0.336

DISCUSSION

The kinetics of EBV DNA load in peripheral blood has been well studied in transplant recipients; however, the kinetics of EBV DNA in IM patients (primary EBV infection) remains unclear. Several investigators have found a negative correlation between EBV DNA copy numbers in serum or peripheral blood samples and sampling time after illness onset (8, 9). The present kinetics data, which was obtained by analysis of serially collected samples, demonstrates a gradual decrease in EBV DNA load after illness onset and supports the previous data. Berger et al. have described the kinetics of serum EBV DNA load in IM patients, and stated that some patients exhibited a slow decline in serum EBV DNA copy numbers after the onset of the illness (9). However, to our knowledge, this is the first report, which demonstrates that the kinetics of EBV DNA copy numbers in PBMCs can be a basis for dividing IM patients into two types, slow regression and rapid regression.

The severity and duration of symptoms varies in IM patients and some authors have previously explored factors associated with clinical features. Viral load has been shown to be correlated with disease severity in several other herpesvirus infections (10, 11). Balfour et al. found a positive correlation between EBV DNA copy numbers in PBMCs and disease severity as determined by a scoring system (12). EBV DNA copy numbers in PBMCs may correlate with the severity of sore throat (13). In contrast, other investigators have reported contrary findings (14). It is well known that primary EBV infection in young children is generally subclinical, the frequency of typical IM patients being relatively high in older children or young adults. However, we observed no statistically significant differences in the quantity of EBV DNA in PBMCs and plasma among different age groups of IM patients (5). Since, as suggested by the present study and previous reports, copy numbers of EBV DNA may be affected by sampling time (8, 9), examination of only one time point is unlikely to clarify any correlations between EBV DNA load and the clinical features of IM patients. Therefore, we looked for any correlations between the kinetics of EBV DNA copy numbers and clinical features or laboratory findings, and indeed observed no statistically significant differences between the slow and rapid regression groups. Our data suggest that the kinetics of EBV DNA load in peripheral blood is not correlated with clinical manifestations or disease severity in IM patients.

Natural killer cells, which play a major role in the innate immune response, may be involved in control of EBV infection (15). Many studies have demonstrated an upregulation of cytokines, including TNF-α, IL-12, and IL-1β, in the sera or tonsillar tissues of IM patients (15–17). As there were significantly higher concentrations of these three cytokines in the rapid than in the slow regression group in the present study, the innate immune response may have an important role in controlling the kinetics of EBV DNA load in IM patients. It has been shown that cytokine gene polymorphisms are associated with susceptibility to, and reactivation of, EBV (18–20). Thus, host genetic factors might be involved in the kinetics of EBV DNA load in IM patients. Further genetic analysis of cytokine genes is necessary to confirm this possibility. Additionally, IP-10 and MIG induced by IFN-γ are important in the control of EBV infection (21, 22); we found significantly higher concentrations of these in the rapid than the slow regression group. These two chemokines may have a critical role in the host immune response in lymphoid tissues of IM patient (23). To our knowledge, this is the first report demonstrating that serum chemokines may be associated with control of viral infection in IM patients.

Herein we have clarified the correlations between the host immune response and the kinetics of EBV DNA load in IM patients. Our data highlight the importance of the cytokine and chemokine responses; however, previous studies have suggested that the cytotoxic T cell response against EBV plays an important role in controlling EBV replication (13). Therefore, further study is needed to elucidate the precise role of the host immune response in control of kinetics of EBV DNA in IM patients.

ACKNOWLEDGMENTS

The authors thank Mrs. Akiko Yoshikawa and Mrs. Chieko Mori for technical support.

DISCLOSURE

The authors do not have any commercial or other associations that might pose a conflict of interest.

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