K. Hayashi

Table 3. Comparison of genomic sequence of MHV variants and their pathogenesis in mice
Variants ofMHV

Tumorigenicity

Splenomegaly

t-RNA-likesequences

M1 through M3Genes
M4

M5 through M12

MHV-68
MHV-72
MHV-76++-+-+-+-++-+++MHV, murine gammaherpesvirus.

no integration of HTLV-II genome (Hayashi etal., 1994). Later a type of oncogenic virus, EBVrelated herpesvirus in Si-IIA cells (Si-IIA-EBV)
was identified, and malignant lymphomas inducedby Si-IIA in Japanese White rabbits as well as
New Zealand White rabbits contained EBV-related
DNA (Hayashi et al., 1995). I also established arabbit lymphoma model via the natural oropharyngeal route with Si-IIA-EBV (Koirala et al., 1997)
as well as rabbit lymphoma induction via transfusion with blood from Si-IIA-EBV-infected rabbits(Koirala et al., 2004). In addition I confirmed thelymphomagenesis of rabbits by another EBV-likeherpesvirus variant from cynomolgus (Cyno-EBV)(Hayashi et al., 1995; Chen et al., 1997). However,intravenous inoculation of an HVMF1-infected cellline producing few virus particles (C54) made seroconversion in one of 10 rabbits and did not induce
rabbit lymphomas. Based on the sequence analysisof these three viruses, these can be considered variant virus each other (Hayashi et al., 1999, 2002;
Hayashi and Akagi, 2000). Therefore, I designatethese three viruses of Si-IIA-EBV, Cyno-EBV andHVMF1 as Cynomolgus-EBV in this paper.
Intravenous or peroral inoculation of CynoEBV in rabbits induced a high rate of lymphomagenesis (77–90%). All of the sera from rabbits inoculated intravenously or perorally with Cynomolgus-EBV-producing cells or EBV-producing B95-8
cells showed increased anti-VCA IgG antibody
titers (× 10–10,240). The autopsy of tumor-bearing
rabbits revealed marked splenomegaly, lymph nodeswelling and/or hepatomegaly with multiple white
nodules. White tumor nodules were less frequently
found in the kidneys and heart. Rarely, multiple
peritoneal and skin metastatic tumors were observed.

A histological examination of rabbit tissues
revealed malignant lymphomas involving manyorgans. All of the involved tissues were classified
as non-Hodgkin’s lymphoma, diffuse, large-cell ordiffuse mixed type (Figs. 3a, b, e and f). Bizarre
giant cells were seen occasionally, admixed withlymphoma cells. Less often, bizarre giant cells
were identified in a non-neoplastic background,mimicking the morphology of Hodgkin’s lymphoma (Figs. 3c and d).
The in situ hybridization studies revealed that
EBER-1 expression was detected in most Cynomolgus-EBV-producing cells and in about 90% casesof Cynomolgus-EBV-induced rabbit lymphoma.
Most of the multinucleated bizarre giant cells
among both neoplastic and non-neoplastic cells
were positive (Figs. 3b and d).
Eleven T-cell lines harboring EBV-related
DNA and EBER-1 expression were establishedfrom Cynomolgus-EBV-induced tumor-bearing
rabbits (Fig. 3g). Type I/II latency of EBV infection was observed in Cynomolgus-EBV-induced
lymphomas and their cell lines. Interestingly, sixof them showed a deletion or translocation of 12q[12q–, 5 cases; t (7p+: 12q–), 1 case]. All cell lines
except one (B6-J130LN) showed tumorigenicity innude mice (Fig. 3h).
Direct sequencing of the three PCR products
revealed that Si-IIA-EBV had about 82 % nucleotide similarity to the human EBV DNA in three regions (BRRF1 and IR1 regions) (Baer et al., 1984).
Si-IIA-EBV had about 92.4% nucleotide similarity
to HVMF1 (Ino et al., 1997). Cyno-EBV DNA has
77% base pair homology to EBV DNA from B95-8
cells and 91% base pair homology to HVMF1 DNA
in the IR1 region (Hayashi et al., 1999). These sequence data indicate that Si-IIA-EBV has higher46EBV-associated diseases and their animal models

bacdfeghFig. 3. Cynomolgus-EBV-induced lymphomas in rabbits. Diffuse large cell lymphoma of the spleen (hematoxylinand eosin stain) (a) and EBER-1 (in situ hybridization) (b). Hodgkin-like lymphomas with EBER-1 expression are
rarely observed (hematoxylin and eosin stain) (c) and EBER-1 (in situ hybridization) (d). Lymphoma cells also infiltrate into the testis (e) and brain (f). Phase-contrast microphotograph of the rabbit lymphoma cell line (g) and its
transplanted tumor in a nude mouse (h).

47

K. Hayashi

sequence homology to HVMF1 and Cyno-EBV
than human EBV from B95-8 suggesting that CynoEBV, Si-IIA-EBV and HVMF1 differ from B958-EBV and may be variants of each other (Hayashiand Akagi, 2000: Ohara et al., 2000).

White rabbits resulted in seroconversion to EBVVCA in 7 of 10 rabbits and 1 of 4 rabbits, respectively. And all 8 seroconverted rabbits developd
T-cell lymphoma within 3 to 9 months after inoculation. Necropsy revealed splenomegaly or hepatomegaly or both in most tumor-bearing animals.
White nodules were frequently found in kidneys,
heart and lungs. Lymph node enlargement and skininvolvement were rarely observed. Histologically,
diffuse mixed lymphoma cells infiltrated involving many organs of rabbits. Viral sequences fromtissue DNA were detected by PCR in all lymphomatous rabbits. Not all cells within the lymphoidinfiltrates expressed EBER-specific viral RNA,and the intensity of the EBV EBER staining variedamong cells of the same tissue. Possibly the lowlevel expression found using EBER was related toa suboptimal sensitivity of this technical approachowing to the incomplete homology of the probe
used because the DNA sequence encoding EBER
from HVMNE is unknown. HVMNE-DNA andEBV-like RNA expression was also detected in two
transformed T-cell lines established from two lymphomatous rabbits. Analysis of one of these T-celllines demonstrated the persistence of HVMNEDNA, expression of an LMP1-like protein, acquisition of interleukin-2 independence, and constitutive
activation of the Jak/STAT pathway. HVMNE infection of rabbits provides a valuable animal modelfor human EBV-associated T-cell lymphoma whereby genetic determinants for T-cell transformationby this EBV-like animal virus can be studied.

Rabbit lymphoma model induced by EBVlike herpesvirus from Macaca arctoides: Herpesvirus Macaca arctoides (HVMA) is an EBVlike herpesvirus isolated from lymphoid cell line ofthe rhesus monkey species Macaca arctoides (Lapinet al., 1985). Both simian T-cell leukemia virus(STLV-1) and HVMA are produced by MAL celllines established from Macaca arctoides. Inoculation of MAL cells in rabbits induced malignantlymphomas and PCR analysis revealed the presence of T-cell leukemia virus-like sequence but not
the presence of EBV-like sequence in rabbit lymphomas (Schatzl et al., 1993). However, Wutzler etal. (1995) demonstrated the association of HVMA
with the etiology of malignant lymphoma of rabbits
inoculated with HVMA by detecting EBER-liketranscripts expression and HVMA-DNA with PCR
in the lymphoma cells. Inoculation of HVMA into
32 rabbits resulted in the seroconversion to EBVVCA and EBV-EA in all infected rabbits and showing symptoms in 16 cases (50%) between 21 and143 days after inoculation, and the development of
17 LPD (13 high-grade non-Hodgkin’s lymphomasand 4 lymphoid hyperplasia). The phenotype ofLPD was not described and latency type of EBV
infection in rabbit LPD could not be determined.
However, these findings suggested that HVMA
caused a symptomatic infection and subsequent
LPD development in rabbits (Wutzler et al., 1995).

Baboon EBV (herpesvirus papio)-inducedrabbit model for EBV-associated fatal LPD withvirus -associated hemophagocytic syndrome(VAHS): Human EBV-associated hemophagocytic
syndrome (EBV-AHS), has a poor prognosis and isoften noted in patients with fatal IM (Mroczek etal., 1987; Okano and Gross, 1996), fatal childhood
with T-cell LPD (Su et al., 1994, 1995; Kikuta,1995), chronic active EBV infection (Yamashitaet al., 1998), and malignant lymphomas (MLs),
particularly EBV-infected T-cell lymphoma (Craiget al., 1992; Su et al., 1993). Patients with HPS
exhibit common clinicopathologic features such asRabbit T-cell lymphoma model induced byEBV-like-herpesvirus from Macaca nemestrina:
Herpesvirus Macaca nemestrina (HVMNE) isa novel EBV-like virus isolated from a Macaca
nemestrina with CD8+ T-cell mycosis fungoidescutaneous T-cell lymphoma (Rivadeneira et al.,1999). A new rabbit T-cell lymphoma model by
HVMNE has been reported (Ferrari et al., 2001).
Intravenous inoculation of HVMNE -infected
T- cells or cell-free HVMNE in New Zealand48

EBV-associated diseases and their animal models

fever, skin lesions, lung infiltrates, hepatosplenomegaly with jaundice and liver dysfunction, pancytopenia and coagulopathy. The liver, spleen, lymph
nodes and bone marrow are usually infiltrated withproliferated florid histiocytes with hemophagocytosis as well as proliferated atypical lymphocytes.
Increased serum levels of many cytokines including soluble IL-2, IL-1, IL-3 and IL-6, macrophagecolony stimulating factor, interferon-γ, prostaglandins and tumor necrosis factor-alpha (TNF- α)have also been reported (Su et al., 1995; Okano andGross, 1996).
Herpesvirus papio (HVP) infection-related
rabbit fatal LPD with VAHS, which is described indetail as in the following, is the first animal modelfor human EBV-fatal LPD with VAHS (Hayashi etal., 2001, 2003a).
An HVP-producing baboon lymphoblastoid
cell line (594S) or cell-free HVP virion pellets
obtained from 594S culture were intravenously inoculated into female New Zealand White rabbits.
Of the 13 rabbits inoculated intravenously withHVP-producing simian 594S cells, 11 (85%) diedof LPD 22 to 105 days after inoculation. LPD wasalso accompanied by VAHS in 9 of these 11 rabbits. Peroral inoculation of cell-free HVP resulted
in viral infection in 3 of 5 rabbits, with 2 of the 3
infected rabbits dying of LPD with VAHS (51–81days). LPD with VAHS was also induced in 7 of
7 rabbits (100%) by intravenous injection of cellfree HVP 21 to 28 days after inoculation. In total,
only 3 infected rabbits remained free of LPD. Twoof the rabbits that showed no seroconversion afterperoral inoculation exhibited no abnormalities.
Increased anti-EBV-VCA IgG antibody titers(× 40–2,560) were detected in all sera from rabbits
inoculated intravenously with 594S (HVP). However, increased anti-VCA-IgG antibodies levels
were found in only 3 of the 5 rabbits inoculated
perorally with cell-free virion pellets. Peripheralblood (PB) examination of some rabbits with LPD
and VAHS revealed elevated GOT (≤ 116 IU/L),GPT (≤ 109 IU/L) and LDH (≤ 1,557 IU/L), andleukocytosis (≤ 21,500/mm3) with mildly increased
levels of atypical lymphocytes (1–10 %). Transient mild leukopenia (3,700–5,400/mm3) was also

found in 4 of the 10 rabbits examined.
Except for anorexia and emaciation, mostrabbits inoculated with HVP appeared physically normal, but showed severe bloody rhinorrhea(Fig. 4a) and dyspnea during the few days before
death. Autopsy of the infected rabbits frequently
revealed pulmonary congestion and edema, often
accompanied with severe hemorrhage of the lungs.
Mild or marked splenomegaly with congestion andhemorrhage (Fig. 4b) was often observed, as well
as dark purple, swollen lymph nodes with hemorrhage (Fig. 4c) and/or hepatomegaly. White nodules were sometimes found in spleen, liver or heartcross-sections. Histological examination of rabbit
tissues revealed mild to severe infiltration of atypical pleomorphic lymphoid cells involving manyorgans. Atypical large or medium-sized lymphoidcells without Hodgkin’s cell-like morphology infiltrated around perivascular areas with a diffuse
or nodular pattern. Apoptotic cells (individual cellnecrosis) accompanied by histiocytes containing
cellular debris were often observed in the atypicalcell-infiltrated lesions. Lymph nodes, spleen, andliver were frequently and markedly involved. Most
involved lymph nodes showed diffuse infiltrationsof atypical lymphoid cells and marked hemophagocytosis in the sinus (Figs. 4d and e). Involved livers
showed severe periportal and sinusoidal infiltrationof atypical lymphoid cells (Figs. 4f and g), which
was often accompanied by central necrosis of thehepatic lobules. Atypical lymphocytes were often
found in the blood vessels. Hemophagocytosis wasalso found in the spleen, bone marrow and thymus.
Six rabbit T-cell lines with IL-2 dependency
were established from 3 of the 5 HVP-infected
Japanese White rabbits. Five of 6 cell lines had thenormal rabbit female karyotype (44, XX), while
one had an abnormal karyotype (Hayashi et al.,2003a). In 18 of 20 LPD cases (90%), EBER-1
expression was detected in virtually all atypicallymphoid cells (Figs. 4e and g). EBER-1-positive
atypical lymphoid cells infiltrated not only theparenchyma and stroma of the various organs, butwere also sometimes demonstrated in the vessels,
lymph nodal sinus or hepatic sinusoid. The six rabbit lymphoid cell lines also expressed EBER-1.

49