Yonago Acta medica 2005;48:41–56

EBV-Associated Diseases in Humans and their Animalin vivo Models: Part IIKazuhiko Hayashi
Division of Molecular Pathology, Department of Microbiology and Pathology, School ofMedicine, 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, Burkitt’s lymphoma
and nasopharyngeal carcinoma are well-known EBV-associated diseases. The range ofEBV-associated diseases has recently expanded to include Hodgkin’s lymphoma, T-celllymphoma, 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 hemophagocyticsyndrome. 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

marmoset (Callithrix jacchus) and the owl monkey(Aotus trivirgatus). The cotton-top tamarin provides an in vivo model for EBV-persistent infection
and EBV-related lymphomagenesis (Niedobitek etal., 1994). The EBV-infected cotton-top tamarins
show a spectrum of responses that varies fromunapparent infection to frank malignant lymphoma (Miller et al., 1977; Rickinson and Kieff,
1996). The cotton-top tamarin with inoculation ofa high transforming dose of EBV developed multiple tumor foci composed of immunoblasts andplasmacytoid cells with the full spectrum of virus
latent genes characteristic of latency type III infection (Cleary et al., 1985). These experimentallyinduced lymphomas are very good for the B-cell

Animal models for EBV infection
and EBV-associated diseases
EBV-associated diseases and their animal models
are tabulated in Table 2. In this review, I will focus
only on in vivo animal models for EBV-related diseases.
Animal models using human EBVMonkey models using human EBVThe host range of EBV is limited to humans
and some new world monkeys (the cotton-toptamarin (Sanguinus oedipus oedipus), the common

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LPD seen in immunocompromised humans. The
common marmoset also provides an in vivo model
for primary and persistent EBV infection, but notfor EBV-related lymphoma. However, the valueof tamarin and marmoset model as a more generalmodel for EBV infections in humans is limited by
the inability to infect these animals via the naturaloropharyngeal route and/or virus persistence inanimals, which do not develop lymphoma (Rickinson and Kieff, 1996). New World primates are
endangered species, rare and so expensive that theyare difficult to use for experiment.

technology (Wilson., 1997). Candidate oncogenes
can be definitively tested and their role in tumor
formation dissected in vivo. In developing transgenic mouse models of EBV-associated diseases,
the mechanism of action of the viral proteins,gleaned from molecular and biochemical analyses,
can be visualized as phenotypic consequence in thewhole organism. For examples, expression of theEBNA-1 or LMP-1 induces B cell lymphoma intransgenic mice (Wilson et al., 1996; Kulwichit etal., 1998).
Animal models using EBV-like herpesvirus(lymphocryptovirus)

SCID mouse model using human EBVSevere combined immunodeficiency disease(SCID) mice with xenografts of human EBV-infected lymphocytes frequently developed oligoclonal or polyclonal multiple foci of EBV-positive human B-cell LPD (Moiser et al., 1998). These SCID
tumor cells had a normal karyotype and showed
latency type III infection with a small minority ofcells expressing lytic viral proteins. Analysis of
EBV clonality revealed that these tumors contained
multiple viral episomes and linear DNA indicating
viral replication. The SCID mouse model therefore
is a convenient in vivo system in which to assess
novel therapeutic regimes directed against EBVassociated B cell LPD (Johannessen and Crawford,1999).

Simian models using naturally infected proper
herpesvirus (Rhesus and Cynomolgus models)
Lymphocryptoviruses (LCVs) are endemic inprimate species and resemble each other in genomic structure and gene organization. Their structuraland nonstructural proteins are frequently antigenically reactive across species (Kieff and Rickinson, 2001). Not only various Old World monkey
species but New World monkeys carry their own
EBV-related LCV (Dillneer et al., 1987; Kieff andRickinson, 2001; Wang et al., 2001). And this isprobably the reason why they were refractory tohuman EBV infection trials. These viruses show
extensive colinear genome homology with EBV,
encode many antigenically related proteins including EBNA-1 and EBNA-2 homologues, and canimmortalize their natural target B cells in vitro but
are not usually associated with any known diseaseof natural host monkeys. Their common evolutionary origin with EBV strongly suggests that the essential features of the virus-host interaction havebeen conserved (Rickinson and Kieff, 1996).
Rhesus monkey provides a new model forprimary and persistent EBV infection. Experimen-The other models using human EBVSome of rabbits orally inoculated with EBV(B95-8) showed EBV infection with continuous
detection of EBV-DNA from peripheral blood by
PCR for 18 weeks and transient rise of antibodies
to EBV but developed no tumors (Koirala et al.,
1997; Chen et al., 1997).
Studies of tumorigenic mechanisms have beenpromoted by the application of transgenic mouse

Abbreviations: AIDS, acquired imunodeficiency syndrome; CTL, cytotoxic T lymphocyte; EBER, EBV-encoded
small RNA; EBNA, EBV-determined nuclear antigen; EBV, Epstein-Barr virus; EBV-AHS, EBV-associated hemophagocytic syndrome; HHV, human herpesvirus; HPS, hemophagocytic syndrome; HTLV, human T lymphotropicvirus; HVMA, Herpesvirus Macaca arctoides; HVMF1, Herpesvirus Macaca fascicularis-1; HVMNE, Herpesvirus
Macaca nemestrina; HVP, Herpesvirus papio; IM, infectious mononucleosis; LCV, lymphocryptvirus; LMP, latentmembrane protein; LPD, lymphoproliferative disease; MHV, murine gammaherpesvirus; SCID, severe combined
immunodeficiency; SIV, simian immunodeficiency virus; VAHS, virus-associated hemophagocytic syndrome; VCA,viral capsid antigen

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EBV-associated diseases and their animal models

tal oral inoculation of rhesus LCV in LCV-naïve
rhesus monkeys resulted in acute and persistent
LCV infection mimicking EBV infection in hu-mans. Acute responses resemble to those seen inhumans infectious mononucleosis with atypicallymphocytosis and activated CD23-positive B cells

Table 2. Animal models of EBV-associated diseases
Animal models
Animals used

Viruses

Diseases and pathology

Similar EBV-associateddiseases of humans

Animal models using human EBVMonkey models
Cotton-topmarmoset
Calithrix
marmoset
Owl monkey

EBV

EBV-associated LPD

PTLD-like lymphoma (B-LPD)

EBV

EBV-associated LPD, mild

PTLD-like lymphoma (B-LPD)

EBV

EBV-associated LPD,
disseminated

PTLD-like lymphoma (B-LPD)

EBV-infected lymphocyte

Transplanted human LPD

PTLD-like lymphoma (B-LPD)

EBV-infected lymphocyte Transplanted human LPD

PTLD-like lymphoma (B-LPD)

EBVMild IMMouse model
SCID mouse

The other animal models
Hamster(newborn)
RabbitAcute infection (mild IM)

Animal models using EBV-like herpesvirus (lymphocryptovirus)
Monkey models using naturally infected proper herpesvirus
Rhesus monkey
Cynomolgus
with SIV
infection

Rhesus-EBV (lymphocryptovirus)
Cynomolgus-EBV(herpesvirus from
Macaca fascicularis)

Acute Rhesus-EBV infection
or IM; oral hairy leukoplakia
Cynomolgus-EBVassociated LPD

Acute EBV infection or IM;
oral hairy leukoplakia
PTLD-like lymphoma (B-LPD)

LPDLPD
Acute infection

PTLD-like lymphoma (B-LPD)
PTLD-like lymphoma (B-LPD)
Acute EBV infection or IM

Cyno-EBV-associatedML (T-cell)
HVMA-associated ML

EBV-associated ML(T-cell)
EBV-associated ML (?)

HVMNE-associatedML (T-cell)
HVP-associatd fatal LPD
with VAHS (T-cell)

EBV-associated ML(T-cell)
EBV-associated fatal LPD
with VAHS (T-LPD)

Mouse models using MHV
Mouse (Balb/C)
Mouse (Balb/C)
Mouse (Balb/C)

MHV-68
MHV-72
MHV-76

Rabbit models using simian EBV-like herpesvirus
RabbitRabbitRabbitRabbitCyno-EBV, Si-IIAEBV (HVMF1)
Herpesvirus from Macaca
arctoides (HVMA)
Herpesvirus from Macaca
nemestrina (HVMNE)
Boboon-EBV [Herpesvirus papio (HVP)]

B-LPD, B-cell LPD; LPD, lymphoproliferative disease; PTLD, post-transplant LPD; T-LPD, T-cell LPD; VAHS,
virus-associated hemophagocytic syndrome.

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K. Hayashi

in peripheral blood with cross-reacting antibodies
to EBNA2 and EBV-VCA. Acute infection wasfollowed by a persistent infection with shedding
virus in saliva and harboring asymptomatic LCVin the peripheral blood. However, without overtimmunosuppression, LCV-related tumors have notdeveloped in this model (Wang, 2001; Wang et al.,2001). Rhesus LCV can infect epithelial cells in
immunosuppressed rhesus macaques and can induce epithelial cell lesions resembling oral hairyleukoplakia in AIDS patients. Electron microscopy, immunohistochemistry and DNA-RNA in situ
hybridization were used to identify the presence ofa lytic rhesus LCV infection in these proliferative,
hyperkeratotic or parakeratotic epithelial cell lesions (Kutok et al., 2004).
An EBV- like her pesvir us (Her pesvir us
Macaca fascicularis -1, HVMF1) isolated fromlymphomas of simian immunodeficiency virus(SIV)-infected Cynomolgus monkeys (Macaca fascicularis) has been identified as a causative agent
for a monkey model of EBV-associated lymphomagenesis in human AIDS (Feichtinger et al., 1992;
Rezikyan et al., 1995). Rhesus monkeys (Macacamulatta) and Cynomolgus monkeys infected witha SIV developed B cell lymphomagenesis at 4%
and 31% incidence, respectively, associated with an
EBV-related simian herpesvirus (Rhesus LCV andHVMF1, respectively), providing a monkey modelfor EBV-associated lymphomagenesis at 3 to 6%incidence in human AIDS (Habis et al., 1999). Of
160 consecutive renal transplants of cynomolgus,
5.6% developed B-cell LPD with EBER expression
28 to 103 days after transplantation (Schmidtko etal., 2002).

trikova et al., 2000). Especially MHV-68 has been
intensively investigated to be used as a mouse model for LPD induced by EBV. Intranasal inoculationof MHV-68 in Balb/c mice induced viral infection
and replication in the lung alveolar epithelia andmononuclear cells and subsequently followed by infectious mononucleosis and/or persistent infection
in murine B cells. Twenty of 220 (9%) persistently
infected mice developed MHV-68-associated LPD
during 3 years observation and the LPD incidenceof MHV- 68 infected mice with Cyclosporin A
treatment increased to 60% (Sunil-Chandra et al.,1994). In situ hybridization revealed the presenceof viral DNA and the expression of viral RNA in
the lymphoid cells of LPD lesions. An MHV-68infected B cell line derived from an LPD lesion
showed tumorigenicity in nude mice (Usherwoodet al., 1996). Atypical lymphocytosis in acutephase of mouse MHV-72 infection, like infectious
mononucleosis in humans and LPD development
in MHV-72-infected Balb/c mice, were reported(Mistrikova and Mrmusova, 1998). Pathology ofMHV-72-infection in CB17 +/+ and CB17 scid/scid
mice was examined (Fig. 2; Oda et al., submitted).
Comparative genomic sequence analysis of MHV
variants and their different pathogenesis wereshown in Table 3 (Macrae et al., 2001; Oda et al.,submitted). Many aspects of MHV-68 or MHV-72
infection in mice are similar to those of human
EBV infection and this is a useful model for thestudy of gammmaherpesvirus infection in vivo.
Rabbit models using simian EBV-like herpesviruses
Rabbit T-cell lymphoma model induced by
Cynomolgus-EBV (herpesvirus from Macacafascularis): I previously established a simian (Cynomolgus monkey) leukocyte cell line (Si-IIA) by
cocultivation with an human T lymphotropic virus(HTLV)-II-producing human CD8+ T cell line(HTLV-IIA) (Miyamoto et al., 1990). Si-IIA cells
immortalize human T cells (Hayashi et al., 1993;
Ohara et al., 1993). During a study on the prevention of HTLV-II infection using Si-IIA, I foundby chance that malignant lymphoma develops inJapanese White rabbits when they were inoculated
intravenously and that these rabbit lymphomas had

Mouse models using murine gamma-herpesvirus
Murine herpesvirus 68 (MHV-68), a murine
gammaherpesvirus, was isolated from a murid rodent, the bank vole in Slovakia (Blaskovic et al.,1980). Seven more isolates similar to MHV-68(MHV-60, MHV-72, MHV-76, MHV-78, MHVSumava, MH V- 4556 a nd MH V-5682) werealso obtained. At least three isolates, MHV-68,
MHV-72 and MHV-Sumava seem to be involvedin malignant neoplasm development in mice (Mis-44EBV-associated diseases and their animal models

acbde

200 nmf: Detection of MHV-DNA in MHV-72-infected mice
PCR for v-cyclin of MHV-68

P72 P68 N

123

4

5

67739 bp –PCR for M1 gene of MHV-68
1 kbp –

P72P68

N1Fig. 2. Pathology of MHV-72-infected mice.
a: Acute bronchopneumonia induced by nasal inoculation of MHV-72 in CB17 +/+ mouse.
b: Malignant lymphoma occurred in the spleen of MHV-72-infected CB17 +/+ mouse.
c: Hemophagocytosis detected in the spleen of MHV-72-infected CB17+/+ mouse.
d & e: Intranuclear inclusions of herpesvirus in the many hepatocytes of MHV-72-infected CB17 scid/scid mouse.
f: Detection of MHV-DNA in MHV-72-infected mouse tissues by PCR using MHV-68 primers.
v- cyclin gene was detected in MHV-72-infected mouse tissues (lanes 1 through 7), MHV-68 (lane P68) andMHV-72 (lane P72). However, M1 gene of MHV-68 was only amplified in the positive control (lane P68) and not inMHV-72 (lane P72) and MHV-72-infected tissues (lane 1). N, the negative control.

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