ying yang ORCID iD: 0000-0003-2294-7249

Feng Gao ORCID iD: 0000-0003-4907-7212

Clinical characteristics of primary and reactivated

Accepted Article
Epstein-Barr virus infection in children

a 6-year observational study

Ying Yang1，Gao Feng2

1
Department of Neurology，The Children’s Hospital, Zhejiang University

School of Medicine, National Clinical Research Center for Child Health,

Hangzhou, 310052, China

2
Department of Neurology，The Children’s Hospital, Zhejiang University

School of Medicine, National Clinical Research Center for Child Health,

Hangzhou, 310052, China

Corresponding author：Yang Ying, 6510124@zju.edu.cn, No.3333 Binsheng

Road, Binjiang District, Hangzhou, Zhejiang, 310052, China

Abstract

Objective: Epstein-Barr virus (EBV) infection occurs commonly in children and presents as

primary or reactivated infection, which are difficult for clinicians to distinguish. This study

investigated the clinical characteristics of the two types of infections. Methods: Children with

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Record. Please cite this article as doi: 10.1002/jmv.26202.

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 detectable plasma EBV-DNA were retrospectively enrolled and divided into primary and

reactivated infection group by EBV-specific antibody. We analyzed the patients’

characteristics, clinical manifestations, complications, inflammatory biomarkers and viral load.

Results: 9.3% of children with reactivation were immunocompromised over the long-term.
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Primary infection mostly appeared as infectious mononucleosis (99.8%), while reactivation

occurred as infectious mononucleosis like disease (65.0%), hemophagocytic syndrome

(22.6%), chronic active EBV infection (5.3%) and lymphoma (3.5%). The incidence of fevers,

cervical lymphoditis, periorbital edema, pharyngotonsillitis, hepatomegaly and splenomegaly

in primary infection were 93.3%，93.0%，51.5%，66.0%，76.2% and 63.9%, respectively; the

incidence of those symptoms in reactivation was 84.0%, 46.9%，15.4%, 18.5%, 18.5%, and

43.3%, respectively. The incidence of digestive, respiratory, cardiovascular，neurological，

hematological，genitourinary complications and multiple serous effusion in primary infection

was 68.8%，18.1%，8.0%，0.8%，2.9%，0.0% and 2.3%; whereas the incidence of these

complications in reactivation was 56.2%，22.5%，14.1%，8.0%，38.9%，0.3% and 19.0%.

Patients with reactivation were more prone to multi-systemic damage. B-cells were lower, and

CD8+ T-cells were higher in primary infection. Viral load was correlated with the level of

different cytokines in primary and reactivated infection. Conclusions: EBV primary infection

often presents as infectious mononucleosis. Reactivated infection affects more

immunocompromised subjects with diverse and complex manifestations. Various

complications are more commonly associated with reactivation as a result of different

inflammatory responses to different types of infection.

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 Keywords: Epstein-Barr virus; reactivation; inflammation

Abbreviations: EBV Epstein-Barr Virus; EBVPI Epstein-Barr Virus Primary

Infection; EBVRI Epstein-Barr Virus Reactivated Infection; VCA Virus Capsid

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Antigen; IgM Immunoglobulin M; IgG Immunoglobulin G; NA Nuclear Antigen;

IM Infectious Mononucleosis; HPS Hemophagocytic Syndrome; CAEBV

Chronic Active Epstein-Barr Virus Infection; EA Early Antigen; IL Interleukin;

TNF Tumor Necrosis Factor; IFN Interferon; ALT Alanine-aminotransferase;

AST Aspartate-aminotransferase

Author Contribution

Author 1 designed the study, collected the data, performed the analysis and

wrote the paper. Author 2 collected the data and performed the analysis.

Acknowledgments

We thank our clinical officer, medical officer and data entry and field staff for

their contributions to this study.

Introduction

Epstein-Barr virus (EBV) is a member of the γ-herpes virus family. EBV infection is one

of the most common infections in humans, and approximately 90% of humans have acquired

EBV infection by the age of 18[1]. It is a major and important pathogen in children all over the

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 world, which is a serious threat to children’s health and brings a heavy burden to patients,

families and society. There are EBV primary infection (EBVPI) (newly established), and EBV

reactivated infection (EBVRI). Although EBVPI and EBVRI are confusing in the way that

they manifest and differ in their pathogenesis, management, and prognosis. For instance,
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EBVPI typically is self-limited, but EBVRI is different. It can be recurrent and associated

with severe complications or poor prognosis in patients with long-term immune deficiency. It

can also become chronic infection, or aggravate other diseases in previous healthy

individuals[2, 3]. EBVRI requires targeted treatments, such as chemical therapy or bone

marrow transplantation, while EBVPI does not[4]. Few studies have focused on the differences

between the two, which is difficult for clinicians to distinguish. This study investigated the

patients’ characteristics, related diseases, clinical manifestations, complications, inflammatory

biomarkers and viral load in order to help clinical doctors to manage the infection caused by

EBV.

Materials and Methods

Samples

This was a retrospective observational study of EBV infection in patients

during the period from January 2012 to December 2017 in the Children’s

Hospital, Zhejiang University School of Medicine, National Clinical Research

Center for Child Health. Patients who met the following criteria were enrolled: (1)

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 <18 years old. (2) diagnosis with EBV infection by positive plasma EBV DNA.

All subjects were divided into EBVPI and EBVRI according to EBV-specific

antibody serology: EBVPI was defined as the presence of Virus Capsid Antigen

(VCA) immunoglobulin M (IgM) and VCA immunoglobulin G(IgG) without

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Nuclear Antigen (NA) IgG. EBVRI was defined as the presence of VCA IgG and

NA IgG with/without VCA IgM[5, 6]. EBV-related diseases in this study included

EBV-related infectious mononucleosis (IM)[7], EBV-related hemophagocytic

syndrome (HPS)[8], chronic active EBV infection (CAEBV)[9], EBV-related

lymphomas[10], IM-like disease, and EBV-related single system damage (such as

EBV-related encephalitis, thrombocytopenia, interstitial pneumonia, hepatis and

myocarditis). (3) rule out those unable to differentiate EBVPI or EBVRI.

Laboratory tests

EBV DNA

Plasma EBV DNA was measured by real-time fluorescence-based

quantitative PCR technique using the EBV DNA kit (Da An Gene Co., LTD. of

Sun Yat-Sen University, China) [11]. The blood samples were collected and

centrifuged at 1000 g at 20℃ for 20 minutes after clotting. Then the plasma was

collected for DNA extraction. The mixture (20 µl of the plasma and 20 µl DNA

extraction solution) was boiled (100℃) for 10 minutes. After that a total of 4 µl of

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 sample was used as the template for EBV qPCR detection(ABI 7500 detection

system). Negative controls and four standards (104, 105, 106 and 107 copies/mL)

were amplified side by side. DNA loads were calculated using standard curve. A

Ct value ≤ 37 (DNA copy number > 500 copies/mL) was considered positive.
Accepted Article

EBV-specific antibodies

EB VCA IgM, VCA IgG, Early Antigen (EA) IgM and NA IgG were

measured by chemiluminescence based on immunoassay (EBV antibodies kit,

Shenzhen YHLO Biotech Co., Ltd, China). EBA antibodies in plasma samples

after incubation with magnetic particles coated with the specific antigen, forming

antigen-antibody complex, and then detected with Acridine conjugated mouse

anti-human IgG. Pre-excitation. Finally, excitation reagents were added to

generate light signals. The test results were expressed in relative luminous

intensity (RLU), proportional to VCA IgM, VCA IgG, EA IgM and NA IgG

levels in the sample.

Cytokines and lymphocyte subsets

Serum interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF) -α,

and interferon (IFN) -γ levels were measured by cytometry using a CBA kit (BD

biosciences, San Jose, CA). CD20+, CD3+, CD4+, CD8+ and

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 CD3-CD16+CD56+ lymphocytes were measured by cytometry using lymphocyte

subsets kit (Beckman Coulter, CA).

Statistical analysis
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Continuous data with normal distribution were expressed as mean ± standard

deviation ( ± S), while those with non-normal distribution were expressed by

median (P50). Categorical data were expressed as percentiles (%). Categorical

variables were compared using the χ2 test and continuous variables using the

two-sided Mann-Whitney U nonparametric test between two groups. Correlations

of viral load with different EBV-related diseases, system damages or cytokines

was analyzed using Spearman rank correlation test. All statistical analyses were

performed using Microsoft Excel 2007 and SPSS Statistics 20.0 software. A p <

0.05 was considered statistically significant.

Results

Patients’ characteristics

A total of 739 patients were enrolled in this study. Among them, 513(69.4%)

had a primary EBV infection and 226(30.6%) had a reactivated EBV infection.

The median age was 3.92 years (P10~P90:1.50~9.25 years) and male to female

ratio was 1.30:1(418:321). The median age in EBVPI was 4.17 years (P10~P90：

1.68~8.12 years), older than that (3.33 years) of EBVRI (P10~P90：1.00~10.43

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 years) (p = 0.004). Among the patients with EBVRI, 21(9.3%) cases were

immunocompromised over a long period of time, due to organ transplantation

and/or chemotherapy (11 cases), nephrotic syndrome (2 cases) and others. No

patients with long-term immunosuppression were found in EBVPI. Some inducing

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factors that produce temporary immune dysfunction were found in EBVRI, such

as previous severe sepsis (22 cases).

EBV-related diseases

EBVPI overwhelmingly manifested as IM (99.8%, 512/513), and HPS only

accounted for 0.6% of the patients (3/513). Two of the three HPS patients had

concomitant IM. EBVRI had diverse diseases including IM-like disease (65.0%,

147/226), CAEBV (5.3%, 12/226), HPS (22.6%, 51/226), lymphoma (3.5%,

8/226), and EBV-related single system damage (6.6%, 15/226). Single system

damage presented as encephalitis (2.2%,5/226), hepatitis (0.9%, 2/226) or

thrombocytopenia (3.5%, 8/226). Among them, 5 cases were CAEBV with HPS, 1

case was CAEBV with lymphoma and 1 case was lymphoma with HPS.

EBV-related infectious mononucleosis like symptoms and complications

EBV-related IM-like symptoms included fevers, cervical lymphoditis,

periorbital edema, pharyngotonsillitis, hepatomegaly, and splenomegaly.

EBV-related complications included: respiratory complications (upper airway

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 obstruction and/or pneumonia), cardiovascular complications (acute myocarditis

and/or atherosclerosis), gastrointestinal complications (hepatitis, acute acalculous

cholecystitis, liver failure and/or splenic rupture), hematological complications

(thrombocytopenia, aplastic anemia, agranulocytosis and/or lymphohistiocytosis),

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neurological complications (facial nerve palsy, Guillain-Barre syndrome and/or

encephalitis), and genitourinary complications (renal dysfunction and/or genital

ulcers).

All typical IM-like symptoms more frequently appeared in EBVPI (Table 1).

Complications occurred in 76.0% (562/739) of patients, 46.2% (342/739) with one

systemic complication, 21.1% (156/739) with two systemic complications, and

8.6% (64/739) with more than two systemic complications. EBVRI

(47.8%,108/226) were more likely accompanied by multi-systemic complications,

compared to EBVPI (21.8%, 112/513) (p<0.001). The most common complication

occurred in digestive system (65.0%, 480/739), and liver dysfunction was the

leading manifestation (97.5%, 468/480). 97.0% (465/468) developed

non-cholestatic hepatitis, 3.0% (14/468) developed cholestatic hepatitis and no

liver failure occurred. EBVRI developed a higher frequency of hematological,

cardiovascular, neurological, genitourinary complications and multiple serious

effusion, compared to EBVPI (Table 2).

Inflammatory biomarkers

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 In peripheral blood cell analysis, a higher frequency of hematopenia was seen

in EBVRI, including reductions of leukocytes, erythrocytes, and platelets.

Leukocyte count, lymphocyte ratio, and atypical lymphocytes were higher in

EBVPI. In an analysis of lymphocyte subsets, B-cells, CD4+ T-cells were lower

Accepted Article

and CD8+T-cells were higher in EBVPI compared to EBVRI (Table 3). A higher

viral load was associated with higher level of cytokines IL-6, IL-10, IFN-γin

primary infection and with higher level of cytokines IL-10, TNF-α, IFN-γin

reactivation (Table 4).

Viral load

Viral load differed significantly among various EBV associated diseases (p <

0.001) (Figure 1), and higher viral load was associated with multi-systemic

damage (p < 0.001) (Figure 2).

Discussion

EBV, also known as human herpes virus 4, is a DNA virus and a member of

the herpes virus family. The EBV lifecycle consists of latent and lytic phases.

Both primary or reactivated infection can trigger the lytic phase. EBVPI initially

takes place in epithelial cells of the oropharynx and nasopharynx. Later, it enters

the affected tissues and infects B-cells. EBV infection becomes latent (the latent

phase) in memory B-cells. Under certain pathological conditions it can start active

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 replication again through reactivation, resulting in cell lysis (lytic phase). EBV

reactivation represents a health challenge to the host.

This investigation consisted of the largest number of cases with EBV primary
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and reactivated infection based on available literature. EBV infections were more

common in male children than female. Most children in our study had primary

EBV infection in their early life (under 4 years old), while it was delayed in

western countries[12]. However, median age with symptomatic EBVRI onset was

younger than symptomatic EBVPI. The likely reason is that young children often

are asymptomatic when primary infection occurs, but it soon becomes latent with

frequent reactivation[13]. Obviously, host immune status was closely related to

EBV infection. Most EBVPI occurred in immunocompetent hosts, while more

EBVRI appeared after immunocompromised events like sepsis, transplant or

chemotherapy[14-16].

EBV has been implicated in a wide range of diseases in children. Most

EBVPI were presented as IM and seldom as HPS, while patients with EBVRI

revealed complex and diverse clinical manifestations. Some patients with EBVRI

can appear as full or partial IM-like symptoms, which is called IM-like disease,

and some would have chronic or recurrent IM-like symptoms that persist for more

than 3 months and become CAEBV. CAEBV is often accompanied by poor

prognosis[17]. In addition, EBVRI is implicated in many subtypes of lymphomas,

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 such as peripheral NK/T cell lymphoma, B-cell lymphomas, extranidal NK/T-cell

lymphoma, Burkitt’s lymphoma and diffuse large B-cell lymphoma[18]. EBV-HPS

frequently occurs upon reactivation, and the prognosis is significantly worse than

those with primary infection[19, 20]. However, we found that some EBVRI patients
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display different clinical manifestations during the disease course, such as

CAEBV with HPS or CAEBV with lymphoma[21]. This suggests that EBV

reactivation causes a wider spectrum of diseases and more complex and variable

clinical manifestations than primary infection. Since it’s more difficult to identify

EBVRI, both careful evaluation and close follow-up are required.

EBVPI and EBVRI may present similar symptoms despite distinct features.

We found all IM-like symptoms (fevers, cervical lymphadenitis, periorbital

edema, pharyngotonsillitis, hepatomegaly and splenomegaly) were more typical

and frequent in EBVPI compared EBVRI. Both EBVPI and EBVRI resulted in

various complications [22]. Digestive system complications were the most

common. It was characterized by an increase in liver enzymes, rarely

cholestasis[23] or liver failure[24]. Unlike EBVPI with elevated single ALT,

EBVRI's liver damage was characterized by simultaneous elevations of ALT and

AST. This indicated that the liver can be one of the main organs affected by

chronic EBV activation. The respiratory system was the second most common

target site of EBV infection, which manifested as airway obstruction or interstitial

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 pneumonia. Lung injury has been reported with high incidence of respiratory

failure or pulmonary fibrosis with a fatal outcome[25]. Neurological complications

were not rare, including direct viral damage and secondary immunity damage with

diverse and non-specific manifestations and good prognosis[26]. In addition, EBV

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infection can cause rare complications of hematological[27], cardiovascular[28], and

genitourinary[29] systems. EBVRI was prone to multi-systemic damage.

All these symptoms and complications are associated with T‐cell activation

and cytokine production[30]. Since the clinical course emerges differently, EBVPI

and EBVRI differed significantly in the level of specific cytokines and

lymphocytes. In primary infection, B-cells are infected first, followed by the

activation of CD8+ T-cells. Activated CD8+ T-cells can destroy virus-infected

cells through cytotoxicity. Infected B-cells undergo necrosis or apoptosis,

resulting in a decrease number of B-cells and an increase in CD8+ T-cells. The

increase in CD8+ T-cells results in a reversal of CD4/CD8 ratio[31]. Activated

T-cells produce various cytokines to cause clinical symptoms. After primary

infection, a normal immune system strictly controls the proliferation of virus to

prevent reactivation, so the body and virus are in a balance. Both CD4+ and CD8+

T-cells are critical in the process of virus reactivation from latency, and their

response to virus are thought to be significantly different between primary and

reactivated infection, though the mechanism is not clear yet[32, 33]. Our hope was to

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 understand this mechanism. The proliferation of CD8+ T-cells was significantly

suppressed during reactivation, suggesting that cytotoxic functions are partially

impaired, which cause large-scale replication of the virus. On the other hand, we

found CD4+ and CD8+ T-cells stimulated by reactivation still produce a large
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number of cytokines, which cause many serious symptoms and complications.

The higher levels of cytokines (including Th1 and Th2 cytokines) were associated

with a higher viral load and cytokine pattern in primary infection was different

from reactivation, indicating that various cytokines produced by T-cells play an

important role in the pathogenesis, which was reported by previous studies[34].

Unfortunately, this study did not have the opportunity to investigate specific

T-cell functions, which will be included in our future studies.

EBV load is a specific marker for some EBV-related diseases[35-37]. An

unusually high viral load might provide a hint for HPS or CAEBV[35]. In addition,

we found that the early higher viral load was associated with more complications.

This is an important finding that may have implications for predicting patient

severity and prognosis. This may be explained by the initial viral load that

determines the level of T-cell response and systemic inflammation. In support of

this relationship, a recent study showed that viral load was significantly higher in

patients with severe symptoms[38].

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 In conclusion, symptomatic EBV infection in children manifests as primary

and reactivated infection. It is recommended that diagnosticians perform both

EBV-DNA and antibody tests in children with suspected EBV infection or with a

high risk for EBV activation. Most EBVPI children are immunocompetent with
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typical IM clinical manifestation. EBVRI frequently follows

immunocompromised events, and presents as diverse and complex clinical

manifestations. Complications may involve different organs in both two

infections, and liver damage is the most common. We first report that hosts have

different inflammatory responses due to two EBV infections. The mechanisms

remain unclear and warrant further study. One limitation of our study is that we

only analyzed clinical characteristics of EBV infection without probing its

regulatory mechanism, which needs to be included in future studies.

Compliance with Ethical Standards

Conflict of Interest: The authors declare that they have no conflict of interest.

Funding: There is no funding source.

Ethical approval：All study protocols were approved by the Ethics Committee of

Children’s Hospital of Zhejiang University School of Medicine

(NO.2018-IRB-079).

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 Informed consent: Informed consent of children and their legal guardians were

waived as a retrospective study.

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Accepted Article

Table 1 EBV-related infectious mononucleosis like symptoms

Primary infection (n=513) Reactivated infection (n=226) p value

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 Frequency count frequency Count

Fevers 93.3% 479 84.0% 190 <0.001

Cervical lymphadenitis 93.0% 477 46.9% 106 <0.001

Accepted Article

Periorbital edema 51.5% 264 15.4% 35 <0.001

Pharynogosilitis 66.0% 339 18.5% 42 <0.001

Hepatomegaly 76.2% 391 52.7% 119 <0.001

Splenomegaly 63.9% 328 43.3% 98 <0.001

Table 2 EBV-related systemic complications

Primary infection (n=513) Reactivated infection (n=226) p value

frequency count Frequency count

digestive complications 68.8% 353 56.2% 127 0.001

respiratory complications 18.1% 93 22.5% 51 0.60

cardiovascular complications 8.0% 41 14.1% 32 0.015

neurological complications 0.8% 4 8.0% 18 <0.001

hematological complications 2.9% 15 38.9% 88 <0.001

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 multiple serous effusion 2.3% 12 19.0% 43 <0.001

genitourinary complications 0.0% 0 0.3% 1 -

P50 (n=347) P10~P90 P50 (n=160) P10~P90

Accepted Article

ALT (U/) 127.0 49.0 ~341.6 141.0 40.0 ~657.4 0.244

AST (U/L) 99.0 56.0~263.2 152.0 61.2 ~ 683.2 <0.001

DB (μmol/L) 1.9 1.0 ~ 5.5 2.9 1.1~ 43.7 <0.001

Table 3 Inflammatory biomarkers in EBV infection

Primary infection Reactivated infection

p value

normal range P50 P10~P90 P50 P10~P90

Leukocyte (*10^9/L) 4.00 - 12.00 15.98 (513) 9.55~25.10 8.3 (225) 1.82 ~22.34 <0.001

lymphocyte ratio (%) 20.00 - 40.00 64.00 (513) 51.00~76.00 47.9 (224) 15.30~75.35 <0.001

Hemoglobin (g/L) 110.00 - 155.00 122.00 (513) 108.00~134.00 115.00 (225) 84.60 ~ 130.00 <0.001

Platelet (*10^9/L) 100.00 - 400.00 196.00 (513) 128.40~197.60 196.00 (225) 27.20 ~396.80 0.284

atypical lymphocyte (%) 0 6.00 (513) 5.00~14.00 0.00 (160) 0 - 8.00 <0.001

IL-2 (μg/dL) 1.10 - 9.80 3.00 (466) 1.80~4.90 3.0 (186) 1.87~5.49 0.814

IL-4 (μg/dL) 0.10 - 3.00 2.70 (466) 1.80~4.00 3.0 (186) 2.00~4.600 0.003

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 IL-6(μg/dL) 1.70 - 16.60 12.70 (466) 4.37~58.47 23.9 (186) 4.54~ 165.49 <0.001

IL-10 (μg/dL) 2.60 - 4.90 16.85 (466) 5.90~41.66 15.75 (186) 3.17~461.86 0.611

TNF-α(μg/dL) 0.10 - 5.20 2.20 (466) 1.40~4.44 2.40 (186) 1.60~5.56 0.038
Accepted Article

IFN-γ(μg/dL) 1.60 - 17.30 15.30 (466) 6.57~41.29 20.2 (186) 2.14~965.55 0.099

CD20 (%) 14.00 - 21.00 4.68 (427) 1.78 ~10.31 10.91 (153) 2.37~30.22 <0.001

CD3 (%) 60.00 - 71.00 84.40 (427) 71.02~92.52 68.4 (153) 34.73~ 87.90 <0.001

CD4 (%) 37.00 - 48.00 14.44 (427) 8.20~24.48 23.06 (153) 7.47~46.17 <0.001

CD8 (%) 16.00 - 21.00 61.22 (427) 38.88~76.41 27.26 (153) 12.22~62.83 <0.001

CD3-CD16+CD56+ (%) 7.00 - 14.00 5.46 (427) 2.62~10.88 6.35 (153) 1.92~17.70 0.034

CD4/CD8 1.9 - 2.9/1 0.24 (427) 0.12~0.55 1.04 (153) 0.17~ 2.39 <0.001

Table 4 Correlations of plasma EBV load and cytokine levels

Primary infection

Spearman correlation coefficient P value

IL-2(μg/dL) 0.067 0.146

IL-4(μg/dL) 0.078 0.093

IL-6(μg/dL) 0.124 0.007

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 IL-10(μg/dL) 0.356 <0.001

TNF-α(μg/dL) 0.026 0.577

IFN-γ(μg/dL) 0.352 <0.001

Accepted Article

Reactivated infection

Spearman correlation coefficient P value

IL-2(μg/dL) 0.071 0.337

IL-4(μg/dL) -0.008 0.912

IL-6(μg/dL) 0.109 0.140

IL-10(μg/dL) 0.487 <0.001

TNF-α(μg/dL) 0.262 <0.001

IFN-γ(μg/dL) 0.485 <0.001

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