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Infectious Transmission of Human T-cell Lymphotropic Virus Type I1 in Rabbits
By G.L. Cockerell, M.G. Weiser, J. Rovnak, B. Wicks-Beard, B. Roberts, A. Post, I.S.Y. Chen, and M.D. Lairmore
To determine the susceptibility of rabbits t o experimental
infection with human T-cell lymphotropic virus t y p e 4 (HTLV11). four separate groups of four weanling rabbits each were
inoculated intravenously with lethally irradiated HTLV-IIinfected human cell lines Mo-T (HTLV-11,-infected
T cells),
WIL-NRA (an Epstein-Barr virus [EBVI-transformed B-lymphoblastoid cell line infected with HTLV-I,),
729pH6neo (an
EBV-transformed lymphoblastoid cell line transfected with a
or G12.1 (HTLV-ll-infected T
molecular clone of HTLV-11,).
cells from a Panamanian Guaymi Indian). Two additional
groups of four rabbits each were similarly inoculated with
control uninfected 729 or HUT 78 cells. Early and persistent
seroconversion t o HTLV-II core antigen p24, as determined
by Western immunoblot, occurred in all HTLV-ll-inoculated
rabbits and was most intense in rabbits inoculated with
G12.1 cells; seroreqctivity t o other HTLV-II gag or env
antigens occurred later, with less intensity, or not in all
inoculated rabbits. Peripheral blood mononuclear cells
(PBMC) and other lymphoid cells from HTLV-ll-inoculated
rabbits produced minimal p24 in vitro, as determined by
enzyme immunosorbent capture assay. Virus was more
readily detected by polymerase chain reaction amplification
of HTLV-II pol sequences; this occurred most frequently in
rabbits inoculated with Mo-T cells, and most frequently in
PBMC as compared with other tissues tested (bone marrow,
brain, and liver). No evidence of disease occurred in HTLV-IIinoculated rabbits observed for as long as 24 weeks. All
control rabbits remained negative for evidence of HTLV-II
infection, as determined by the same procedures. These
results provide the first evidence of HTLV-II infection in a
species other than humans, and demonstrate the usefulness
of the rabbit as an animal model t o study the biologic
response t o different isolates of this human retrovirus.
0 1991by The American Society of Hematology.
T
tions, 36 of 39 and 21 of 23 HTLV-infected individuals were
confirmed by polymerase chain reaction (PCR) to be
infected with HTLV-II.'4*'5In addition, HTLV-I1 infection
has been documented in a population of Guaymi Indians in
PanamaI6; this represents the first description of HTLV-I1
infection outside of traditional risk groups.
The known pathogenicity of HTLV-I and the high
seroprevalence of HTLV-I1 in certain risk groups justifies
additional study of these human retroviruses. This should
include the identification of appropriate auimal models to
study the pathogenesis, prevention, and treatment of the
infection and disease. Previous work has shown that rabbits
can be infected with HTLV-I by the intravenous, intraperitoneal, and oral routes with HTLV-I-infected lymphoid
cells of human or rabbit origin."-'' In addition, Miyamoto et
a1 recently demonstrated the in vitro transformation of
rabbit leukocytes cocultured with an HTLV-11-infected
human T-cell line." However, to date, infection with
HTLV-I1 in vivo has not been reported in any species other
than humans. The purpose of the present study was to
determine if rabbits were susceptible to infection with
HTLV-I1 in vivo, and to determine if different HTLV-I1
isolates caused different biologic response patterns in this
animal model similar to emerging findings with HTLV-I
(Lairmore et al, submitted for publication, 1991).
HE HUMAN T-CELL lymphotropic virus (HTLV)
family, including both HTLV types I and 11, as well as
simian T-cell leukemia virus (STLV) and bovine leukemia
virus (BLV), comprise a group of oncovirinae that shares
structural and functional characteristics and biologic effects
distinct from all other mammalian and avian retroviruses.'4
HTLV-I and BLV are the causative agents of lymphoproliferative disorders referred to as adult T-cell leukemialymphoma (ATLL)' and as enzootic bovine lymphoma
(EBL)6 in humans and cattle, respectively. The role of
STLV in leukemogenesis in primates is less clear.' HTLV-I
is also associated epidemiologically with a neurodegenerative disorder termed HTLV-I-associated myelopathy
(HAM)'or tropical spastic paraparesis (TSP)9 in humans.
In contrast, HTLV-I1 has not been linked to a distinct
clinical syndrome in humans, and only occasional isolates of
this virus have been made.'"."
Limited systematic studies of the seroprevalence of
HTLV in the United States have been performed, but such
studies are complicated by the fact that enzyme immunoassays, the most widely used screening tests for HTLV
antibodies, do not differentiate between infection with
HTLV-I and HTLV-II.'2*'3However, recent evidence suggests that infection with HTLV-I1 may be more widespread
than initially suspected, particularly in intravenous drug
users (IVDU); in two separate studies of IVDU popula-
MATERIALS AND METHODS
From the Department of Pathology, Colorado State University, Fort
Collins, CO; Retrovirus Disease Branch, Centers for Disease Control,
Atlanta, GA; and the Department of Microbiology and Immunology
and Department of Medicine, UCLA School of Medicine, Los Angeles,
CA.
Submitted February 11,1991; accepted May 17, 1991.
Address reprint requests to G.L. Cockerell, D M , PhD, Depament
of Pathology, Colorado State University, Fort Collins, CO 80523.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section 1734 sole4 to
indicate this fact.
0 1991 by The American Society of Hematology.
0006-4971I91 l7806-0019$3.00/0
1532
Rabbits and HTLV-II-infected and control cell inocula. Weanling (8-week-old) specific-pathogen-free New Zealand White rabbits were obtained from a commercial rabbitry (Western Oregon
Rabbit Co, Philomath, OR). Groups of rabbits were inoculated
intravenously with 1.5 x lo7to 10 x lo7HTLV-11-infected cells or
uninfected cells as shown in Table 1. HTLV-11-infected cells were
88% to 98% infected, as determined by fluorescent antibody assay
using an HTLV-1/11 anti-p24 monoclonal antibody (MoAb)." In
addition, soluble p24 antigen was quantified in 3-day culture
supernatants (1 x 106 cellslml) of each cell line (except Mo-T)
using an antigen capture enzyme immunosorbent assay (Coulter
Immunology, Hialeah, FL). The range of p24 detected was 255 to
375 ng/mL. Control uninfected cells were negative by the same
techniques. All cells were lethally irradiated (55 Gy) before
inoculation and effectiveness of the irradiation was verified by
Blood, Vol78, No 6 (September 15), 1991: pp 1532-1537
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HTLV-II INFECTION OF RABBITS
1533
Table 1. Inocula for induction of HTLV-I1 Infection in Rabbits
Dose
(~107)
Cellular
Inocula
(no. of
rabbits)
WIL-NRA
1.5 (4)
729pH6neo 1.5 (4)
G12.1
1.5 (4)
Mo-T
10 (4)
729
1.5 (4)
HUT78
1.5 (2)
10 (2)
Origin of Cellular Inocula
An EBV-transformedhuman B-lymphoblastoid cell line (WIL-2)infected with HTLVI, by cocultivationwith T cells from patient NRA with a T-cell variant of hairy cell
leukemia"
An EBV-transformed human lymphoblastoid
cell line (729)transfected with a molecular
clone of HTLV-II,,"
HTLV-ll-infected T cells from a Panamanian
Guaymi Indian'6
HTLV-ll,,,-infected T-lymphoblastoidcell
line from patient Mo with a T-cell variant
of hairy cell leukemia'"
Non-HTLV-infected EBV-transformed human B-lymphoblastoidcell line (used as
control for WIL-NRA and 729pH6neo-infected cells)
Non-HTLV-II-infected human T-lympho.
blastoid cell line (used as control for
G12.1 and Mo-T-infected cells)
failure of the cells to grow in culture. Infectivity of the inocula was
verified by transfer of infection after coculture with normal rabbit
peripheral blood mononuclear cells (PBMC).
Detection of anti-HTLV-IZ antibodies. Sera (1: 100 dilution)
were tested by Western immunoblot assay (WIB) as previously
de~cribed"~'~
using a commercially prepared HTLV-II,,-infected
cell lysate (Hillcrest Biologicals, Cypress, CA) as target antigen.
Detection of HTLV-II antigens. PBMC were collected from all
rabbits at 4, 12, and 24 weeks postinoculation (PI), and single cell
suspensions of spleen, bone marrow, and mesenteric lymph node
were prepared from rabbits killed at each of these intervals. Cells
were cultured at a concentration of 1 x 106/mLin RPMI 1640with
10% fetal bovine serum, 100 U/mL penicillin, and 100 pg/mL
streptomycin, 2 mmol/L glutamine, 5 pg/mL phytohemagglutinin
(PHA-M; Difco Labs, Detroit, MI), and 10% human interleukin-2
(IL-2;Advanced Biotechnologies Inc, Silver Spring, MD). Aliquots
of culture supernatant were collected at 3, 7, and 14 days and
tested for the presence of HTLV-I1 antigens by enzyme immunosorbent capture assay.I6 Briefly, supernatant was added to wells of
microtiter plates precoated with a murine anti-HTLV-1/11 p24
MoAb (Coulter Immunology). Retention of p24 by the capture
antibody was subsequently detected by the addition of biotinylated
human anti-HTLV-1/11 antiserum, followed by a strepavidinhorseradish peroxidase complex and chromagen. Absorbance values were determined and compared with a standard curve prepared with known amounts of core protein p24 in the same assay.
Detection of HTLV-II provirus. Serial samples of PBMC and
other tissues collected at necropsy were assayed for HTLV-I1 by
PCR as previously described" with minor modifications, using
primers and probe specific for the HTLV-I1 pol region?' Briefly,
each PCR amplification was performed using 1 pg genomic DNA
(approximately 150,000 cells) in a 100 +L reaction volume. The
amplification consisted of 34 repetitive three-step cycles with the
following conditions: 25°C to 95T, and then 2 minutes each at
95"C, SYC, and 72°C per cycle in a thermal cycler (Perkin-Elmer
Cetus, Norwalk, CT). The amplified products were electrophoresed on 1.8% agarose gels, transferred to nylon membranes,
and hybridized with a "P-labeled oligonucleotide probe.
RESULTS
Serologic and clinicopathologic responses to HTL V-II.
All HTLV-11-inoculated rabbits produced antibodies against
HTLV-I1 within 2 to 4 weeks PI and remained persistently
seropositive over the 12- to 24-week period of study. All
control-inoculated rabbits remained seronegative (Table
2). The earliest and strongest HTLV-I1 seroreactivity occurred against p24 antigen in rabbits inoculated with each
HTLV-I1 isolate. WIB-detectable seroreactivity to other
HTLV-I1 gag (p21, pr53) and env (gp46) antigens occurred
later, with less intensity, or not in all inoculated rabbits
(Table 2 and Fig 1). Reactivity to gp61/68 was not evident
by WIB. The time of onset, intensity, and pattern of
seroreactivity varied with the HTLV-11-infected cell line
used for inoculation. The earliest and most intense reactivity occurred in rabbits inoculated with G12.1 cells (Fig 1).
No evidence of disease was observed in HTLV-IIinoculated rabbits studied through 24 weeks PI, as evidenced by differences in weight gains, hematologic profiles,
or the gross or microscopic appearance of tissues between
HTLV-11-inoculated and control-inoculated rabbits.
HTLV-11 antigen production from cultured cells. There
was no clear difference in HTLV p24 Antigen present in
supernatants collected after 3,7, or 14 days of culture. Low
levels of antigen were"detected only in rabbits inoculated
with G12.1 cells; supernatants of PBMC from two of four
rabbits tested at 4 weeks PI, and of spleen cells from one of
two rabbits each tested at 4 weeks and 12 weeks PI
contained 10 to 12 pg/mL HTLV p24 (data not shown).
Levels of HTLV p24 antigen in other culture supernatants
from other rabbits inoculated with other HTLV-11-infected
cells or control cells were below cutoff levels.
Detection of HTLV-IIprovirus in tissues. A summary of
the detection of PCR-amplified HTLV-I1pol sequences in
tissues of rabbits is shown in Table 3 and a representative
Southern blot of the amplified products is shown in Fig 2.
The PCR-amplified pol product was most frequently detected in PBMC from rabbits inoculated with Mo-T cells
(three of four rabbits), and was distributed in multiple
tissues of these rabbits (two of three bone marrows tested,
one of four brains tested, and two of four livers tested).
Interestingly, the most extensive PCR-amplifiedpol reactivity was derived from one rabbit (R841) among those
inoculated with 729pH6neo cells containing a molecular
clone of HTLV-II,, (Fig 2).
DISCUSSION
The results of this study show the infectious transmission
of HTLV-I1 in rabbits and represent the first evidence of
HTLV-I1 infection in vivo in a species other than humans.
Further, the results suggest that rabbits infected with
different isolates of HTLV-I1 express different biologic
response patterns, which may be due at least in part to
heterogeneity amongst the viral isolates. Finally, the demonstration of the in vivo infectivity of a molecular clone of
HTLV-I1 provides a mechanism to map the viral genetic
determinants responsible for different biologic responses in
rabbits.
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1534
COCKERELL ET AL
Table 2. Anti-HTLV-ll Humoral Immune Response (determined by WlB) in HTLV-ll-Inoculated Rabbits
Cellular
Inocula*
WIL-NRA
729pH6neo
G12.1
Mo-T
729
HUT 78
HTLV-II
Antigen
P21
P24
pr53
gP46
P21
P24
pr53
gp46
P21
P24
pr53
9P46
P21
P24
pr53
9P46
P21
P24
pr53
9P46
P21
~ 2 4
pr53
gp46
Weeks PI
0
014t
014
014
014
014
014
014
014
014
014
014
014
014
014
014
014
012
012
012
012
013
01 1
013
013
1
014
014
014
114
2
014
414
014
314
011
011
Oil
Oil
3
014
414
214
214
Oil
011
011
011
4
1I 4
414
214
214
1I 4
314
314
214
214
414
214
414
014
414
214
214
012
012
012
012
012
012
012
012
6
8
12
013
313
113
113
112
212
212
212
112
212
212
112
212
212
212
212
013
313
113
213
112
212
212
212
212
212
212
212
212
212
212
212
013
313
213
113
01 1
01 1
01 1
01 1
Oil
01 1
01 1
011
011
011
011
01 1
01 1
Oil
011
011
011
Oil
011
011
16
20
24
012
212
112
112
012
212
112
212
012
212
112
112
011
011
011
011
01 1
01 1
01 1
01 1
'Cellular inocula as per Table 1.
tNumber of positive rabbitslnumber of rabbits tested. Open data points indicate not determined.
In vivo infectivity was evidenced by an early (as early as 2
weeks PI) and persistent (as late as 24 weeks PI) antiHTLV antibody response and demonstration of viral antigens or amplifiable proviral sequences in tissues of inoculated rabbits. Similar to findings in HTLV-infected
h ~ m a n s , ' the
~ . ~earliest and most intense serologic reactivity occurred against p24 antigen as determined by WIB.
Seroreactivity to env (gp46) antigens was also detectable,
despite the difficulties in using the WIB technique to detect
envelope antibodies due to the expected loss of envelope
viral components during antigen purification." The presence of antibodies to both p24 and gp46 in our rabbits met
the criteria established by the US Public Health Service for
serologic confirmation of HTLV infe~tion.'~
As compared with similar studies with HTLV-1'' (Lairmore et al, submitted for publication, 1991), the HTLV-I1
isolates tested appear to be less infectious and to replicate
less efficiently in rabbits. Only low levels of HTLV-I1
antigen were detected in supernatants of short-term cultures of lymphoid cells, and only from 50% of rabbits
inoculated with G12.1 cells. Since these studies were
conducted, we have discovered that the sensitivity of the
antigen detection technique can be increased by coculturing infected cells with normal mitogen-stimulated rabbit
lymphoblasts. This modification of the technique should
increase the value of data derived in future studies. Gene
amplification by PCR provided a greater ability to detect
the presence of virus in tissue. Of the tissues tested
(including PBMC, bone marrow, brain, and liver), provirus
was most readily detected in PBMC. These results parallel
those of Miyamoto et al, in which HTLV-I1 were demonstrated by PCR, but not by indirect fluorescence, in in vitro
HTLV-11-transformed rabbit leukocytes.*"
No clinicopathologic evidence of disease was detected in
HTLV-11-infected rabbits observed for as long as 24 weeks
PI in this study, similar to results with HTLV-11-infected
rabbits." The absence of disease in HTLV-infected rabbits
parallels the situation that occurs in the vast majority of
HTLV-infected humans.26It is possible that as increased
numbers of HTLV-infected rabbits are studied for extended periods, disease may become apparent. Furthermore, it is possible that other viral isolates or cofactors are
required for the development of disease in HTLV-infected
rabbits, again paralleling the potential importance of these
considerations in humans.
The biologic response pattern of HTLV-11-infected rabbits varied depending on the viral isolate used for inoculation. This variation included the time of onset and pattern
of antiviral antibody response and the frequency, distribution, and intensity of PCR amplifiable proviral sequences in
tissues. The earliest seroconversion and the most intense
seroreactivity occurred in rabbits infected with G12.1 cells,
while provirus was most frequently detected in all tissues
tested from rabbits infected with Mo-T cells. It is unlikely
that these differences were due to differences in amount of
virus administered, because all inocula contained approximately the same number of infected cells. However, the
results may be explained by heterogeneity among the
isolates, including properties such as replication competency, cell/tissue tropism, immunogenicity, or copy number
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HTLV-II iNFECTlON OF RABBITS
pr53
-
SP46
-
p24
p21
0
1
2 3 4
L
'
1535
812
6
0
4
812
0
4
812
0
4
812
4
0
812
0
2
3
61624
ULu
-
R35
R841
R843
R845
R839
R33
Mo-T
729pH6neo
WILNRA
G12.1
729
HUT 78
Fig 1. Representative WlBs illustratingreroreactivity of rabbits inoculatedwith different HTLV-ll-infected cells (Mo-T, 729pH6neo. WIL-NRA,
and G12.1) or control uninfected cells (729 and Hut 78) as a function of weeks PI (shown at bottom of individual strips). Location of HTLV-II
antigens is shown at the left margin.
per infected cell. To provide further support for this
supposition, additional studies will need to be performed
using the same cell typc infected with different HTLV-I1
isolates.
We have reported similar differences in the biological
response pattern of rabbits infected with different isolates
of HTLV-I, but we have becn unable to correlate these
differences with disease expression in patients from which
the isolates were derived (Lairmore et al. submitted for
publication, 1991). To date, few structural differences have
been described between HTLV-I isolates derived from
patients with ATLL and those with HAM/TSP; however,
this remains an incompletely studied and a potentially
important mcchanism for explaining the variable disease
outcome in HTLV-infected patients.
Infectious molecular clones will be required for cxpcrimcnts to unambiguously map the genetic determinants
responsible for spccific biologic activities. It is important
Table 3. Detection of Provirus by PCR in HTLV-ll-lnoculated Rabbks
PBMC (weeks PI1
Cellular
Inocula.
4
12
WiL-NRA
729pH6neo
G12.1
Mo-T
729
HUT70
014t
114
114
214
012
014
112
112
012
212
011
4
12
012
01 1
01 1
01 1
01 1
011
012
112
012
111
01 1
Liver (weeks PI)
Brain lweeks PI)
Bone Marrow (weeks PI)
24
24
111
4
12
012
012
012
011
01 1
011
012
112
012
011
'Cellular inocula as per Table 1 .
tNumber of positive rabbitslnumberof rabbits tested. Open data points indicate not determined.
011
24
112
4
12
24
012
012
112
011
01 1
011
012
112
012
111
112
011
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1536
COCKERELL ET AL
A
B
C
&
- 143 bp
IL
PBMC
u
u
Br
Liv
PBMC
EM
R34
Br
R35
Liv
EM
Br
PBMC
Liv
R36
I
Br
R841
I
Mo-T
EM
729pH6WO
Liv
ld3 id4
Mo-T
HUT
78
Fig 2 Representatbe hybridizationsof PCR-ampltfied H N V - I I provirus in tissues from rabbit. using oligonucleotide primer pairs and probes
specific for HTLV-IIpol sequences.Total genomic DNA was extracted from tissue samples and 1 pg of DNA (equivalentto approximately150,OOO
cells) was used in each PCR amplification. (A) Tissues (PSMC; Br, brain; EM, bone marrow; Liv, liver) from three rabbits (R34, R35. and R36)
inoculatedwith HTLV-ll-infected Mo-T cells, and assayed at either 12 weeks PI (R36).or 24 weeks PI (R34 and R35). (E) Tissues (seedesignations
for A) from a rabbit (R841)inoculatedwith 729pH6neo cells containing a molecular clone of HTLV-II, and assayed at 12 weeks PI. (C) Positive
control HTLV-II-infected Mo-T cells (10 and 10 ‘ dilutions of DNA extracts) and negative control Hut 78 cells. The location of the 143-bp
sequence correspondingto the amplified HTLV-II pol gene is shown at the right margin.
’
and encouraging to notc, thcrcforc. that thc 729pH6nco
ccll linc, which contains a molecular clonc dcrivcd from thc
Mo-T isolatc of HTLV-11, was infectious for rabbits.
Expcrimcnts similar to prcvious in vitro studies with dclction mutants or mutagcnizcd HTLV-I1,,,:7.:” can now bc
pcrformcd in vivo to furthcr clucidatc thc rolc of spccific
viral gcnc scgmcnts in viral rcplication and cellular transformation.
ACKNOWLEDGMENT
The authors express their appreciation to Dr Gary Toedter,
Coulter Immunology, Hialeah, FL, for providing the HTLV-I/II
antigen capture enzyme immunoahsorhent kits.
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HTLV-II INFECTION OF RABBITS
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From www.bloodjournal.org by guest on April 28, 2017. For personal use only.
1991 78: 1532-1537
Infectious transmission of human T-cell lymphotropic virus type II in
rabbits
GL Cockerell, MG Weiser, J Rovnak, B Wicks-Beard, B Roberts, A Post, IS Chen and MD
Lairmore
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