Yonago Acta medica 2005;48:1–6

EBV-Associated Diseases in Humans and their Animalin vivo Models: Part I
Kazuhiko Hayashi
Division of Molecular Pathology, Department of Microbiology and Pathology, School of
Medicine, Tottori University Faculty of Medicine, Yonago 683-8503 Japan
Epstein-Barr virus (EBV) is one of human herpesviruses and a member of the gamma
herpesvirus family (lymphocryptovirus). Infectious mononucleosis, Burkittʼs lymphoma
and nasopharyngeal carcinoma are well-known EBV-associated diseases. The range ofEBV-associated diseases has recently expanded to include Hodgkinʼs lymphoma, T-cell
lymphoma, pyothorax-associated or methotrexate-associated B-cell lymphoma, primaryeffusion lymphoma and lymphoepithelioma-like carcinoma of the stomach, thymus andsalivary gland, lymphoproliferative disorders (LPDs) or leiomyosarcomas from immunocompromized host, oral hairy leukoplakia and EBV-associated hemophagocytic
syndrome. Animal models of human EBV-associated diseases are essential to elucidate
the pathogenesis of EBV-infection and EBV-associated diseases. However, only several
reports on the animal models of EBV infection have been reported. Here I review thesummary of EBV-associated diseases in humans and those previous animal models using
EBV or EBV-like herpesviruses and describe some details on our two newly developed
rabbit models of LPD induced by simian EBV-like viruses and a mouse model with murine gammaherpesvirus. These animal models are useful and inexpensive alternative
experimental model systems for studying the biology and pathogenesis of EBV, and prophylactic and therapeutic regimens.
Key words: animal model; EBV-associated disease; human; lymphocryptovirus

the important subject of such investigation for thelast 40 years. EBV preferentially infects human
B cells, T cells, natural killer (NK) cells, epithelial cells and smooth muscle cells (Rickinson andKieff, 2001). Latent EBV infection occurs in theoropharyngeal epithelium, where EBV virions are
replicated and released from the epithelial cells tosaliva. The EBV-infected cells express a different array of EBV-associated antigens dependingon lytic or latent infection (Baer et al., 1984; Rowe

EBV-associated diseases in humans
Epstein-Barr virus (EBV) is one of eight known
human herpesviruses (HHVs) and a member of the
gamma herpesvirus family (lymphocryptovirus).
EBV was the first human tumor virus identified
from cultured lymphoblasts of Burkittʼs lymphoma(Epstein et al., 1964), and its potential role as a
causative agent of EBV-associated tumors has been

Abbreviations: AIDS, acquired imunodeficiency syndrome; CAEBV, chronic active EBV infection; CTL, cytotoxic
T lymphocyte; EA, early antigen; EBNA, EBV-determined nuclear antigens; EBV, Epstein-Barr virus; EBV-AHS,
EBV-associated hemophagocytic syndrome; HHV, human herpesvirus; IM, infectious mononucleosis; LMP, latentmembrane proteins; LP, leader protein; LPD, lymphoproliferative disease; MHV, murine gammahersesvirus; NK,natural killer; PAL, pyothorax-associated lymphoma; VCA, viral capsid antigen

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K. Hayashi

et al., 1992), and these viral antigens are targetedby EBV-specific cytotoxic T lymphocytes (CTLs)(Catalina et al., 2001). The CTL responses to
EBV infections induce a variety of inflammatory
systemic symptoms. On the other hand, the lackof CTLs such as in patients with congenital immunodeficiencies or acquired immunodeficiency syndrome (AIDS) or in recipients receiving a potent
immunosuppressant, allows EBV-infected cells to
proliferate and result in the development of lethal
lymphoproliferative diseases (LPDs). EBV is classically associated with infectious mononucleosis(IM), Burkittʼs lymphoma in equatorial Africa andnasopharyngeal carcinoma (Rickinson and Kieff,
2001). The range of EBV-associated diseases has
recently expanded to include oral hairy leukoplakia,
leiomyosarcoma from AIDS patients, Hodgkinʼslymphoma, T-cell lymphoma, Ki-1 lymphoma,pyothorax-associated B-cell lymphoma, methotrexate-associated B-cell lymphoma, primary effusion
lymphoma, LPDs of primary and secondary immunodeficiency, and lymphoepithelioma-like carcinoma of the stomach, thymus, lung and salivary
gland (Weiss et al., 1989; Chang et al., 1992; Weiss
and Chang, 1996; Anagnostopoulos and Hummel,
1996; Kawa, 2000; Iwatuski et al., 2004) (Table 1,
Fig. 1).

class II molecules to induce cell membrane fusion
in B cells (Molesworth et al., 2000). Binding of
the gp42 molecule to HLA class II is essential forvirus entry into B cells. Following an incubation period of 2 to 7 weeks, EBV-infected B cells increasein number during acute IM. The EBV-infected B
cells, however, are quickly abrogated by cellularimmune responses mediated by NK cells, activatedT cells and antibody-dependent cell-mediated cytotoxicity (Rickinson and Moss, 1997). CD8+, HLADR+ activated T cells increase in peripheral blood,
and are defined as “mononucleosis” by hemograms
when systemic symptoms manifest. During IM as
primary EBV infection, EBV is capable of expressing all viral antigens of lytic cycle: immediate
early, early and late proteins, and all antigens of latent infection genes: six EBV-determined nuclear
antigens (EBNAs) consisting of EBNA-1, -2, -3A,
-3B, -3C and -leader protein (LP), and three latentmembrane proteins (LMPs), including LMP-1, -2A
and -2B (latency type III, Table 1). All viral antigens of lytic cycle, all EBNAs and LMPs, except
for EBNA-1, are target molecules for EBV-specificcytotoxic T cells. Therefore, CTLs direted to these
EBV antigens suppress the EBV-infected cells in
immunocompetent hosts.
EBV-associated hemophagocytic syndrome
Hemophagocytic syndrome is an unusual syndrome
characterized by fever, splenomegaly, jaundice,pancytopenia, disseminated intravascular coagulation, and features of increased hemophagocytic
macrophages in the bone marrow and other tissues(Imashuku, 2002). EBV-associated hemophagocytic syndrome (EBV-AHS) may be associatedwith acute IM, and EBV-associated LPD, lymphomas, cancers and autoimmune diseases, and commonly occurs in children and adolescents in Asia,
but is rarely seen in Western countries. Laboratory
examinations reveal pancytopenia, liver dysfunction, coagulopathy, elevated levels of lactate dehydrogenase, ferritin, β2-microglobulin, and serum
cytokines including IFN-γ, IL-6, IL-10, sIL-2R,
sFas and Fas L (Imashuku, 2002). These cytokines
increased in the process of CTL responses directedagainst EBV-infected CD8+ T cells may induce he-Primary EBV infection and its associateddiseases
Infectious mononucleosis
Acute IM is clinically characterized by fever,lymphadenopathy, tonsillitis, pharyngolaryngitits
and hepatosplenomegaly, and increased IgM and
IgG antibodies to EBV-viral capsid antigens (VCAs)
and early antigens (EAs). Acute IM is fairly common in the United States and Western Europe,
where a primary infection often occurs duringadolescence. Asymptomatic primary infections arecommon in Asia and developing countries because
primary EBV infections occur early in life. EBVvirions bearing gp350/220 infect B cells via CD21
(CR2) or a receptor for C3d, and form an episomal
EBV in the nucleus (Fingeroth et al., 1984). A
complex of gp85(gH)/gp25(gL)/gp42 binds to HLA

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EBV-associated diseases and their animal models

mophagocytosis by activated macrophages. Latent
EBV infection is also detected in other lymphocyte

subsets of CD4+ T cells, CD16+ NK cells andCD20+ B cells (Kasahara et al., 2001).

Table 1. Comparative overview of EBV-associated diseases (tumors) in humans and their compatible animal models
Type ofinfection ordiseaseEBV-associateddiseases

Proposedcellof origin

EBV latent
gene expression

Latency
type

Animalmodel

EBNA-1+, -2+, -3A+,-3B+, -3C+, -LP+,
LMP-1+, -2+
EBNA-1+, (LMP-1+),
LMP-2+
EBNA-1+, -2+, -3A+,-3B+, -3C+, -LP+,
LMP-1+, -2+
EBNA-1+, -2+, -3A+,-3B+, -3C+, -LP+,
LMP-1+, -2+

III
(with
lytic cells)
I/II
III
(with
lytic cells)
III
(with
lytic
cells)+(monkey,mice)+(rabbit)–+(monkey,mice,
scid mice)

EBNA-1+, LMP-1+, -2+

II–

Primary
infectionIM

B cellPrimary
infection
Chronic
infection/
LPD
Chronic
infection/
LPD

EBV-AHS/fatal IM(T-LPD)
X-linked LPD

T cellChronic
infection/
LPD
Lymphoma
Lymphoma
Lymphoma(co-infection
with HHV-8)
Lymphoma
Lymphoma

PTLD-like lymphoma
B lympho(B-LPD) from immunoblast
compromised host(congenital immunodeficiency,transplantation, AIDS)
Chronic active EBV
T cell or
infection
NK cell
Burkittʼs lymphoma
Pyothorax-associatedlymphoma
Primary effusion
lymphoma
Methotraxate-associatedlymphoma (LPD)
Hodgkinʼs lymphoma

Lymphoma/leukemia
Lymphoma/leukemia
Carcinoma

T-cell lymphoma

Carcinoma

Nasopharyngeal
carcinoma
Oral hairy leukoplakia

Virus replicative lesion
SarcomaB cellNasal T/NK cell
lymphoma
Gastric carcinoma

Leiomyosarcoma from
immunocompromisedhost

B cell
EBNA-1+(centroblast)
B cell
EBNA-2+, LMP-1+

I

–

III

–

B cell

EBNA-1+, (LMP-1+),

I/II

–

B cell(LMP-1+)

I/II ?

–

B cell
EBNA-1+, LMP-1+, -2+(centrocyte)
T cell
EBNA-1+, (LMP-1+),
LMP-2+
T/NK cell
EBNA-1+, (LMP-1+),
LMP-2+
Epithelial
EBNA-1+, LMP-2+
cell
Squamous
EBNA-1+, (LMP-1+),
cellLMP-2+Squamous
EBNA-1+, -2+, -3A+,
cell-3B+, -3C+, -LP+,
LMP-1+, -2+
SmoothEBNA-1+, -2+, -3A+,
muscle-3B+, -3C+, -LP+,
LMP-1+, -2+

II

–

I/II
I/II

+
(rabbit)
–

I/II

–

I/II

–

III
(with
lytic cells)
III+(monkey)–This table was modified from the tables in the textbook (Rickinson and Kieff, 2001).
EBV, Epstein-Barr virus; EBV-AHS, EBV-sassociated hemophagocytic syndrome; HHV-8, human herpesvirus-8;
IM, infectious mononucleosis; LPD, lymphoproliferative disease; PTLD, post-transplant lymphoproliferative disease.

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K. Hayashi

against EBV antigens, which may allow the survivalof EBV-infected NK or T cells.

EBV-associated LPD or leiomyosarcomasin immunocompromized indivisuals

EBV latent infection and tumor development
Most EBV-associated LPDs are of B cell lineage,
but T cell neoplasms and Hodgkinʼs lymphoma
may occur. Latent EBV infection plays a pivotal
role in the occurrence of African Burkittʼs lymphoma, pyothorax-associated lymphomas (PALs),
Hodgkinʼs lymphoma (Figs. 1a and b), primary effusion lymphoma induced by HHV-8 coinfection,and various types of B cell lymphomas (Iwatsuki etal., 2004). The association of latent EBV infection
with NK/T cell lymphomas is less common than B
cell lymphomas. Extranodal NK/T-cell lymphoma,
nasal type is more prevalent in Asia, Mexico andCentral and South America, and a disease characterized by histological features of angiocentricand angiodestructive infiltration of lymphoma cellswith severe ulceration or necrosis in nasal cavity.
All those LPD except PAL have latency type I or II,
whereas latency type III is detected in PAL. PAL
is considered to have a localized immunodeficiency
in pyothorax lesions.
EBV-associated carcinomas such as nasopharyngeal carcinoma and stomach carcinoma (Figs.
1c and d) show latency type I/II. Entry of EBV
into epithelial cells that do not express CD21 or
HLA class II is mediated by gp85(gH)/gp25(gL)complexes without gp42 (Borza and Hutt-Fletcher,2002). The restriction of EBV gene expression
such as latency type I (or II) showing only EBNA-1(or LMPs) allows EBV-infected cells to evade immune surveillance, and persists throughout latent
infection. EBNA-1 is essential for replicating EBV
episomes during latency by binding to OriP, a cisacting element of the EBV genome, and by promoting the replication of viral episomes by host cell
DNA polymerase. LMP-1 shows oncogene activity
with the upregulation of cellular gene expression invarious cell types, and prevents apoptosis by the
induction of bcl-2 (Rickinson and Kieff, 2001).
EBV-AHS, CAEBV and NK/T cell lymphomas are common in Asia (Iwatsuki et al., 2004).
Since EBV gene expression is restricted in these
disorders (latency II), only LMPs expressed by

In patients with congenital immunodeficiencies
or AIDS or in recipients of organ transplantaions
receiving a potent immunosuppressant, the lack or
suppression of CTLs caused the EBV-associatedB-cell LPD with latency type III infection of EBV.
EBV is also involved in patients with methotrexateassociated LPD. Leiomyosarcomas may also develop in the immunocompromized hosts, althoughmechanism of EBV infection to smooth muscles
has not been clarified (Rickinson and Kieff, 2001).
Chronic and latent EBV infection and theirassociated diseases
X-linked lymphoproliferative syndrome (Duncan
disease)In patients with X-linked lymphoproliferative syndrome, an inherited immunodeficiency characterized by increased susceptibility to EBV and B-cell
LPD (Nichols et al., 1998), the presence of smalldeletions and mutations in DSHP/SH2DIA/SLAMassociated protein (SAP) gene was detected.
Chronic active EBV infection
Chronic active EBV infection (CAEBV) is a disease of CD4+ T-cell or NK cell LPD characterized
by chronic or recurrent infectious mononucleosis–
like symptoms persisting over a long time and byabnormally high titers of anti-EBV antibodies andincreased levels of EBV-DNA in the peripheral
blood (Okano, 1991). CAEBV is also characterized
by a high mortality and high morbidity with lifethreatening complications, such as virus-associatedhemophagocytic synrome, interstitial pneumonia
and malignant lymphomas several years after disease onset. Patients with T-cell CAEBV had ashorter survival time than those with NK-cell typeof disease (Kimura et al., 2001). Latency type II infection of EBV expressing EBNA-1 and LMPs proteins is demonstrated in cases with CAEBV. However, patients with CAEBV may have congenital or
acquired immunological defects in CTL responses

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EBV-associated diseases and their animal models

a

b

c

d

Fig. 1. Representative EBV-associated tumors in humans.
a: Hodgkinʼs lymphoma, hematoxylin and eosin stain.
b: EBER-1+ Hodgkin and Reed-Sternberg cells observed in Hodgkinʼs lymphoma. EBV-EBER-1 ISH.
c: Gastric adenocarcinoma, hematoxylin and eosin stain.
d: EBER-1 expression in adenocarcinoma of stomach. EBV-EBER-1 ISH.

the EBV-infected cells are targeted by host CTLs.
Despite the absence of overt immunodeficiency,
EBV-infected cells are insufficiently abrogated by
CTLs in these disorders. Therefore, selective immunological defects to LMPs as genetic predisposition can be considered in those patients, including
an HLA-restricted low response of CTLs to LMPs,
immunological tolerance to LMPs and selective deletion of LMP-specific CTLs.
The release of virokines such as vIL-10 andthe down-regulation of cell adhesion molecules are
additional strategies for EBV-infected cells withlatency type I/II to evade the host immune system.
vIL-10 inhibits the synthesis of IFN-γ from lymphocytes and NK cells, and suppresses IFN-γ-mediatedcellular events such as the up-regulation of the MHC
class I expression and CTL responses. Low levels

of intercellular adhesion molecule 1 and leukocyte
function-associated antigen 3 expression are associated with an impaired ability to interact with
EBV-specific CTL (Iwatsuki et al., 2004).
Most EBV-associated tumors arise with a very
long latency in long-term EBV carriers. This suggests the multistep oncogenesis through malignant
transformation from a single cell within the EBVinfected pool (Rickinson and Kieff, 2001). EBV
may require certain risk factors to induce malignancy in humans. These risk factors are immunologic risk factor, genetic risk factors such as racial
predisposition and personal predisposition, andenvironmental risk factors like Euphorbia tirucalli
and malarial infection for African Burkittʼs lymphoma (Osato, 1998). The translocation of c-myc
proto-oncogene and mutation or deletion of genes

5K. Hayashi

like p53 are needed for the development of the mostEBV-associated tumors in addition to EBV infection.
Despite intensive investigations on the roleof EBV infection in the pathogenesis of EBVassociated tumors, a causal relationship between
EBV and these tumors has not been establishedexcept for LPD arising in immunosuppressed individuals. Therefore, animal models of human EBVassociated diseases are essential to elucidate thepathogenesis of EBV-infection and EBV-associateddiseases.

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Received and accepted January 17, 2005
Corresponding author: Kazuhiko Hayashi, MD

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