Download Virological and pathological features of mice infected with murine

2347
Journal of General Virology (1992), 73, 2347 2356. Printed in Great Britain
Virological and pathological features of mice infected with murine
gammaherpesvirus 68
N. P. Sunil-Chandra,* S. Efstathiou,t J. Arno and A. A. Nash
Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K.
The primary infection of BALB/c mice with murine
herpesvirus 68 (MHV-68) was investigated. When the
virus was introduced intranasally, the lung was the
main tissue infected, the virus being associated with
alveolar epithelium and mononuclear cells. A productive infection lasted for 10 days, after which viral DNA
could be detected by in situ hybridization up to 30 days
after infection. At that time lymphoproliferative accumulations were also observed in the lung, with
formation of germinal centres. Virus could also be
recovered from the heart, kidney, adrenal gland and
spleen during the primary infection. In addition, the
spleen appeared to be the major site of virus persistence, with latently infected cells detected up to 90 days
post-infection. During the primary infection, there was
atrophy of the thymus and spleen of clinically sick
animals. In contrast, lymphoproliferative responses,
typified by splenomegaly, were frequently seen in
asymptomatic animals. The pattern of infection observed in MHV-68-infected mice is similar to that seen
in infectious mononucleosis of man following EpsteinBarr virus infection. The model described in this paper
may prove to be useful in studying natural gammaherpesvirus infections of man and domestic animals.
Introduction
valuable for studying the oncogenic potential of EBV
and for vaccine development (Epstein et al., 1985), but
the route of infection used is not the natural one and the
outcome of infection does not mimic that observed in
man.
We are interested in developing an amenable animal
model with which to study permissive natural infection
and latency of gammaherpesviruses. In this paper we
present our initial studies on the pathogenesis of murine
herpesvirus 68 (MHV-68). This virus was originally
isolated from Clethrionomys glareolus (bank vole) in
Czechoslovakia (Blaskovic et al., 1980). Based on the
c.p.e, observed in infected BHK-21 and rabbit lung cell
lines, and on the virion architecture observed by electron
microscopy of ultrathin sections of infected rabbit
embryo fibroblasts, this virus was classified as a member
of the herpesvirus family (Blaskovic et al., 1980;
Ciampor et al., 1981). More recently, analysis of the viral
genome has revealed that MHV-68 is closely related to
the gammaherpesviruses of primates, EBV and herpesvirus saimiri (HVS) (Efstathiou et al., 1990a, b). Studies
carried out in newborn mice with an outbred background
have revealed a virus-induced pneumonia and viraemia
during which virus can be isolated from many tissues
(Rajcani et al., 1985). In this paper, we show that MHV68 infection of BALB/c mice has pathogenic features in
common with those observed in acute EBV infection of
man, and that lung and lymphoid tissues are the major
sites of pathology and virus persistence.
Herpesviruses are linear dsDNA viruses of eukaryotes
which, on the basis of their distinct biological properties,
are divided into three major subgroups: the alpha-, betaand gammaherpesviruses (Roizman, 1982; Honess,
1984; Minson, 1989). The gammaherpesviruses, typified
by Epstein-Barr virus (EBV), have the capacity to
establish a latent infection within their target lymphocyte population, can induce a lymphoproliferative
disease in the infected host and can efficiently immortalize lymphocytes infected in vitro. Although we know a
great deal about the relationship between EBV and B
cells in vitro, and the immune response to EBV has been
well studied (Rickinson et al., 1989), studies of gammaherpesvirus infections have been limited almost entirely
to clinically apparent EBV infection in man and
experimental inoculation of non-human primates with
either EBV or related simian viruses. Studies of acute
infection with EBV are limited to infectious mononucleosis patients, who present some weeks after
infection has occurred and in whom the pathogenesis
may differ from the subclinical infection experienced by
the majority of the population (Rickinson et al., 1985).
The cotton top marmoset is susceptible to EBV-induced
lymphoproliferative disease and this model has proved
]-Present address: Herpesvirus Laboratory, Medical Virology,
Institute of Medicaland Veterinary Science, Adelaide, Australia.
0001-0946 © 1992 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Tue, 02 May 2017 18:34:59
2348
N. P. Sunil-Chandra and others
Methods
Mice. Female BALB/c mice were obtained from Bantin & Kingman
and were infected at 3 to 4 weeks of age.
Viruses and cells. Virus working stocks were prepared by infection of
BHK-21 cells at a low multiplicity (0-1 p.f.u./cell) with MHV-68 clone
G2.4 (Blaskovic et al., 1980; Efstathiou et al., 1990a, b). Virus
suspensions in Eagle's MEM were clarified by centrifugation, and the
supernatant was dispensed in 0.2 ml volumes and stored at - 7 0 °C
until required. Infectious virus was measured by plaque titration using
BHK-21 cells grown in Eagle's MEM containing 10~ (v/v) tryptose
phosphate broth and 10 % (v/v) newborn calf serum at 37 °C in 5 ~ CO2
for 4 days.
Intranasal and intravenous infection o f mice. Groups of 3- to 4-week-old
female mice were inoculated intranasally or intravenously with 4 x 105
p.f.u. MHV-68. Approximately 40p.1 of virus was administered
intranasally to lightly anaesthetized (ether anaesthesia) mice. For
intravenous inoculation, 0.1 ml of the virus dilution was injected into
the tail vein.
Assay of infectious virus. Mice were killed at different times following
infection by injection of Euthatal BP (Vet) intraperitoneally. Blood
samples were obtained from the retro-orbital plexus, and each mouse
was perfused with PBS (20 ml per mouse). The lung, heart, kidney,
liver, spleen, thymus, two mesenteric lymph nodes, adrenal glands,
brain and trigeminal ganglia were removed and stored at - 7 0 °C until
required. Blood clot and sera were obtained from 100 ktl of blood.
Specimens were homogenized separately in 2 ml Eagle's MEM
supplemented with 10~ (v/v) tryptose phosphate broth and 10~ (v/v)
calf serum. The presence of infectious virus was determined by assay on
BHK-21 cells, on which plaques could be detected after 3 to 4 days.
Co-cultivation assay for virus during acute and persistent infection.
Spleen, thymus, lymph nodes and peripheral blood cells were used for
co-cultivation. Cells were obtained from each of these tissues and
suspended in RPMI 1640 containing 20~ (v/v) foetal calf serum (FCS)
(Hunt, 1987). In the case of peripheral blood, total blood cells were
obtained from 100 lal blood immediately after bleeding. In addition, at
90 days post-infection, peripheral blood lymphocytes were obtained by
Ficoll gradient purification for the co-cultivation assay. For the nonlymphoid organs, 1 to 3 mm pieces of tissue were co-cultivated with
BHK-21 cells. Lymphoid cells or tissue explants were co-cultivated
with 2 x 106 BHK-21 cells in RPMI 1640 containing 20~ (v/v) FCS at
37 °C in 5 ~ CO2 for 5 days. The monolayers were fixed and stained,
and the number of plaques were counted. The results were recorded as
numbers of infectious centres per organ or tissue at each time.
Preparation of hyperimmune serum. MHV-68 was grown in RK13
cells; cell-associated virus was disrupted by sonication and the virus
suspension was clarified by centrifugation to remove cell debris. This
preparation was inoculated intramuscularly into rabbits in Freund's
complete adjuvant. Booster injections in Freund's incomplete adjuvant
were given to these rabbits at 2 and 3 week intervals. The sera obtained
from the final bleed were tested for the presence of antibody by
immunofluorescence staining of MHV-68-infected BHK-21 cells. A
1:200 dilution of hyperimmune rabbit serum resulted in specific
staining of virus antigen. For the detection of virus antigen in tissue
sections of infected organs, a 1 : 250 dilution of final bleed rabbit serum
gave optimal specific staining by the indirect immunoperoxidase
technique. Hyperimmune sera were also raised in mice by using a
similar technique.
Histopathological and immunohistochemical studies. Heart, kidney,
lung, liver, spleen, thymus, mesenteric lymph nodes and adrenal gland
were carefully removed from animals at various times after infection
and immediately fixed in 10~ buffered formal saline. Tissues were
embedded in paraffin and 5 ~tm sections were prepared for histopathological examination. Sections were stained using haematoxylin and
eosin, and indirect immunoperoxidase antibody labelling was used to
detect virus antigen. Tissue sections were treated with 0-1 ~ trypsin for
5 min to avoid problems associated with over-fixation of tissue
specimens in formalin. Non-specific binding of antibodies was
minimized by the use of Tris-buffered saline as the diluent and rinsing,
and by the addition of a blocking step which included 5 % normal goat
serum and 5 ~ BSA. Endogenous peroxidase activity was minimized by
immersing the slides in a solution of 0.75% hydrogen peroxide in
methanol for 30 min before trypsin treatment of the tissue sections.
Immunoperoxidase staining with an avidin/biotin detection system
was performed using the Vectastain ABC Kit (Vector Laboratories).
Detection of MHV-68 DNA in tissue sections. Sections from several
tissues taken at different times after infection were prepared as
described above and cut onto poly-L-lysine-coated slides. The method
for detecting viral DNA was a modification of the technique described
by Fazakerley et al. (1991). Briefly, this involved treating tissue sections
with proteinase K, followed by prehybridization (1 h at 37 °C) and then
hybridization with a 1.2 kb DNA probe (boiled for 5 min and rapidly
chilled on ice before addition to the hybridization solution) from the
terminal region of the MHV-68 genome (Efstathiou et at., 1990a, b).
Before the overnight (18 h) hybridization stage, the tissue sections
containing DNA probe were heated to 80 °C for 10 min. The sections
were then washed twice for 15 min with 2 x SSC at room temperature,
twice for 15 min with 0.1 x SSC at room temperature, once for 10 min
with 0.1 × SSC preheated to 60 °C and kept at 37 °C, followed by a
15 min wash with 2 x SSC at room temperature. The sections were then
dehydrated via a series of graded alcohols, air-dried, and then dipped in
photographic emulsion (prepared in a darkroom by adding 20 ml of
Amersham LM-1 emulsion to 20 ml of 0-66 M-ammonium acetate at
42 °C), allowed to dry at room temperature in a light-tight box for 2 h
and autoradiographed for 1 to 4 weeks at 4 °C. The slides were
developed (Ilford Phenisol developer) in a darkroom at 15 °C and fixed,
counterstained with haematoxylin and eosin, dehydrated and mounted
with DePeX.
Results
Determination of infectious virus titres from various
organs during acute infection
Two groups of 25 mice each were inoculated either
intranasally or intravenously. Of the intranasally infected mice, 48 ~ developed clinical signs and symptoms 7 to
9 days post-infection. Severe clinical disease was
characterized by ruffled fur, hunched back, inactivity,
severe weakness and emaciation. Mice showing mild
clinical signs and symptoms recovered at 10 to 12 days,
whereas severely sick mice died during the same period
following intranasal infection. None of the intravenously
infected mice developed a severe clinical illness or died
during this period, indicating that infection by the
intranasal route is more efficient in producing disease
than intravenous administration of virus.
Three mice from each of the intranasally or intravenously inoculated groups were killed on days 1, 3, 5, 10 and
30 post-infection, and samples of lung, spleen, thymus,
mesenteric lymph nodes, peripheral blood, liver, heart,
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Tue, 02 May 2017 18:34:59
MHV-68 infection of mice
2349
Table 1. Virus titres in various organs* of BALB/c mice infected intranasally with MHV-68
Infectious virus titre (log~0 p.f.u./organ)J
Time
post-infection
(days)
Thymus
Mesenteric
lymph node
<1
<0§
<0
1.7
<1
<1
<1
0.9
0.5
<0
0.5
<0
<0
<0
<0
Lung
Spleen
1
4-7
3
5
10
30
6.7
4.7
<1
<1
Whole:~
blood
Adrenal
gland
Heart
Kidney
<0
<1
<1
<0
<0
<0
0.5
<0
<1
<1
5.3
<1
<1
<1
5-1
<1
0.1
0.7
<0
<0
* Infectious virus could not be detected in liver, serum, brain or trigeminal ganglia.
t Mean infectious virus titre for three mice at each time following inoculation of 4 x l0 s p.f.u, of virus/mouse.
$ One-hundred microlitres of whole blood was used.
§ <0, Indicates that no virus was detectable. The limit of detection for adrenal gland, ganglia, blood, thymus and
lymph node was 1 p.f.u./organ; liver, 100 p.f.u.; other organs, 10 p.f.u.
Table 2. Virus titres in various organs* of BALB,/c mice infected intravenously with MHV-68
Infectious virus titre (log10 p.f.u./organ)t
Time
post-infection
(days)
Lung
Spleen
1
<1
<1
3
5
10
30
3-1
2.5
2.1
<1
3.4
2.6
<1
<1
Thymus
Mesenteric
lymph node
Whole:~
blood
Heart
Kidney
Adrenal
gland
Liver
<0§
0.8
0.8
<0
<0
<0
1.3
<0
<0
<0
<0
<0
<0
<0
<0
2.0
3-0
1-8
5-3
<1
<1
<1
<1
5.3
<l
1.9
2-9
4-0
<0
<0
4-6
<2
<2
<2
<2
* In the course of the experiment, 0.3 and 0.7 loglo p.f.u, virus were detected in the trigeminal ganglia and brain
respectively (at 5 days post-infection) of one of 15 mice sampled.
t Mean infectious virus titre for one of three mice at each time following inoculation of 4 x 105 p.f.u, of virus/mouse.
:~One-hundred microlitres of whole blood was used.
§ <0, Indicates that no virus was detectable. The limit of detection for adrenal gland, ganglia, blood, thymus and
lymph node was 1 p.f.u./organ; liver, 100 p.f.u.; other organs, 10 p.f.u.
k i d n e y , b r a i n a n d t r i g e m i n a l g a n g l i a were r e m o v e d a n d
infectious virus was t i t r a t e d ( T a b l e s 1 a n d 2). I n a s e c o n d
e x p e r i m e n t , 70 B A L B / c m i c e were i n f e c t e d i n t r a n a s a l l y
w i t h 4 × 105 p.f.u. M H V - 6 8 a n d at d a i l y i n t e r v a l s up to
10 days, a n d on d a y s 15 a n d 20 five a n i m a l s were killed
a n d v a r i o u s o r g a n s were collected for infectious virus
assay. F o l l o w i n g i n t r a n a s a l inoculation, high titres o f
infectious virus could be d e t e c t e d in lung tissue b e t w e e n
1 a n d 7 days post-infection, a n d t h e r e a f t e r d e c l i n e d to
u n d e t e c t a b l e levels b e t w e e n days 10 a n d 15 (Fig. 1 a n d
T a b l e 1). T h e increase in virus titre b e t w e e n d a y s 1 a n d 3
i n d i c a t e s active r e p l i c a t i o n o f the initial M H V - 6 8
i n o c u l u m in this tissue. A l t h o u g h c l e a r a n c e o f infectious
virus was o b s e r v e d in the lung at 10 d a y s post-infection,
5-3 a n d 5.1 log~o p.f.u, o f virus was d e t e c t e d in t h e h e a r t
a n d k i d n e y o f i n f e c t e d a n i m a l s respectively at this t i m e
( T a b l e 1). H o w e v e r , the d e t e c t i o n o f infectious virus
f r o m h e a r t a n d k i d n e y is n o t a c o n s i s t e n t feature. L o w
titres o f infectious virus could be d e t e c t e d s p o r a d i c a l l y in
the m a j o r l y m p h o i d o r g a n s (spleen, t h y m u s a n d m e s e n teric l y m p h nodes), a d r e n a l g l a n d a n d whole b l o o d
d u r i n g the course o f the e x p e r i m e n t . O u r i n a b i l i t y to
d e t e c t infectious virus in the b r a i n or t r i g e m i n a l g a n g l i a
d u r i n g the acute stage o f infection, a n d the a b s e n c e o f
neurological disease, suggest t h a t M H V - 6 8 is n o t
n e u r o i n v a s i v e following i n t r a n a s a l inoculation.
A s i m i l a r d i s t r i b u t i o n o f infectious virus was o b s e r v e d
in the tissues o f a n i m a l s i n f e c t e d i n t r a v e n o u s l y ( T a b l e 2),
a l t h o u g h i n v o l v e m e n t o f the liver was a p p a r e n t 3 d a y s
p o s t - i n f e c t i o n a n d h i g h e r infectious virus titres were
d e t e c t e d in b o t h the m a j o r l y m p h o i d o r g a n s a n d the
a d r e n a l gland. Since i n t r a n a s a l i n f e c t i o n p r o v e d m o r e
efficient in the g e n e r a t i o n o f clinical d i s e a s e a n d
r e p r e s e n t s a m o r e n a t u r a l infectious route t h a n intravenous a d m i n i s t r a t i o n o f virus it was c h o s e n as the
m o d e l for further study.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Tue, 02 May 2017 18:34:59
N. P. Sunil-Chandra and others
2350
Day 3
Day 5
Day 10
(a)
~6
&
/
~5
~4
!
~ 3
0
.~
2
(b)
w
0
10
Time post-infection (days)
20
Fig. 1. Virus replication in mice during acute MHV-68 infection.
Three- to 4-week-old female BALB/c mice were inoculated with 4 x
l0 s p.f.u, virus intranasally. Mean infectious virus titre/organ + S.D.
for five mice/group is shown for each time for lung (H) and spleen (.).
The limit of detection was 10 p.f.u./organ.
Gross pathological and histopathological changes
Thirty female BALB/c mice were infected intranasally
with 4 × 105 p.f.u, of MHV-68. Three mice were killed at
3, 5, 30 and 170 days post-infection. In addition, six mice,
including three clinically normal but infected and three
showing signs of clinical disease, were killed 10 days
post-infection. Tissues were examined for gross pathological changes before fixation in formal buffered saline.
Haematoxylin- and eosin-stained sections were used to
study histopathologicat changes, and immunoperoxidase
specificity was used to study the location and distribution
of virus antigen.
Gross pathological and histopathological changes in
lymphoid organs were a major feature of MHV-68
infection of BALB/c mice. Animals suffering from
severe clinical symptoms 10 days post-infection exhibited marked splenic and thymic atrophy. No infectious
virus could be detected in these organs at this time (Table
1). In contrast, animals showing no clinical signs
exhibited marked splenomegaly, and moderate mesenteric lymph node and thymic enlargement (Fig. 2). The
cause of the splenic atrophy was lymphoid cell depletion;
the atrophic thymus was attributable to loss of thymocytes (Fig. 3). The splenomegaly associated with the
clinically inapparent infection was characterized by an
increase in the number of germinal centres (Fig. 3).
In the lungs, a dramatic peribronchiolar, perivascular
and interstitial infiltration of lymphoid cells occurred.
Necrosis within the cellular infiltrate was observed as
early as 3 days post-infection, which is consistent with
the lung being a major site of virus replication during
Fig. 2. Gross pathologicalchanges of the spleen followingacute MHV68 infection. Three- ta.4rweek-oldBALB/c mice were inoculated with 4
x l0 s p.f.u, virus intranasally~ Haematoxylin- and eosin-stained,
formalin-fixed longitudinal sections of spleen dissected at 3, 5 and 10
days post-infection are shown to demonstrate relative splenic enlargement or atrophy during acute infection. Clinically sick mice developed
atrophy of the spleen (a); splenomegaly was seen in some clinically
normal mice at 10 days post-infection (b). For reference, a normal 4- to
5-week-old BALB/c mouse spleen was similar in size to that shown for
day 3 post-infection. Bar marker represents 5 mm.
acute infection (Fig. 4, Table 1). At 5 days post-infection,
an exudate of cellular debris was seen in the bronchiolar
lumen and the infiltration of lymphoid cells in the
peribronchiolar, perivascular and interstitial areas was
more extensive. Resolution of the inflammation began at
10 days post-infection in both clinically normal and sick
animals. This is consistent with the clearance of
infectious virus at this stage. However, in the lungs of
mice which had recovered from the primary infection,
localized areas of lymphoid infiltrate persisted at these
same sites, both at 30 and even 170 days post-infection.
In places, germinal centre formation was observed (Fig.
4e). In addition, during and after the acute infection
occasional small clusters of lymphoid cells were seen in
the liver and kidney of the majority of mice.
Immunohistochemical studies of virus antigen distribution
in the tissues of infected mice
Virus antigen was readily detected in the lung during
acute infection (Fig. 5). Foci of positive cells were
observed in the peribronchiolar and perivascular cellular
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Tue, 02 May 2017 18:34:59
MHV-68 infection of mice
2351
Fig. 3. Histopathology of spleen and thymus at 10 days after MHV-68 infection. (a) Atrophied spleen of a clinicallysick infected mouse
showing marked lymphoid cell depletion. WP, White pulp; RP; red pulp. (b) Spleen from a clinicallyinapparent infection. Note the size
of the spleen (splenomegaly) and the increased number of germinal centres. WP, White pulp; RP, red pulp. (c) Atrophied thymus of a
clinicallysick mouse showing almost complete depletion of thymocytes in the thymic cortex and a dense cellular infiltrate in the medulla
with interspersed phagocytic macrophages (P). M, Thymic medulla; C, thymic cortex. (d) Thymus of a clinically normal infected
mouse. The distribution of lymphoid cells is similar to that in uninfected mice (not shown). C, Thymic cortex composed of densely
packed thymocytes; M, thymic medulla, which is paler with fewer thymocytes and more conspicuous epithelial cells. Bar markers
represent (a) and (b) 0.5 mm, (c) and (d) 0.25 mm.
infiltrates at 3 d a y s post-infection. By 5 days, e n l a r g e d
e p i t h e l i a l cells lining t h e alveoli a n d large m o n o n u c l e a r
cells w i t h i n the cellular infiltrates were a n t i g e n - p o s i t i v e .
W h a t a p p e a r e d to be l y m p h o c y t e s w i t h i n those infilt r a t e s were o n t h e whole n e g a t i v e , b u t a few ceils d i d
a p p e a r to c o n t a i n a n t i g e n (Fig. 5), as d i d o c c a s i o n a l
h e p a t o c y t e s a n d m o n o n u c l e a r cells w i t h i n t h e spleen.
T h e p r e s e n c e o f strong s t a i n i n g o f the nucleus a n d also
a p p a r e n t l y o f the c y t o p l a s m o f b o t h e p i t h e l i a l a n d
m o n o n u c l e a r cells strongly suggests t h a t a c t i v e transla-
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Tue, 02 May 2017 18:34:59
2352
N. P. Sunil-Chandra and others
Table 3. Recovery of MHV-68 from primary and secondary lymphoid tissues by a
co-cultivation assay
Infectiouscentres/organ*
Time
post-infection
Mouse
Mesenteric
Peripheral
(days)
no.
Spleen
Thymus
lymph node
blood1"
1
2
1
2
1
2
30
240
300
400
150
200
2
l
0
1
0
4
0
0
0
0
0
0
0
0
0
0
0
3
1
2
3
620
254
178
ND~
ND
ND
ND
ND
ND
ND
ND
ND
!
2
3
4
5
220
0
40
260
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Experiment 1
3
5
10
Experiment 2
36
Experiment 3
90
0
* Number of infectious centres from the entire organ from each mouse.
t In the case of peripheral blood, 100 lal whole blood was used.
"~ND, Not determined.
tion of viral proteins was taking place during the acute
infection (Fig. 5). Immunostaining of the columnar
epithelium of the bronchioles was negative at every
stage. At 10 days post-infection, the amount of viral
antigen recognized by this method was much reduced, a
finding which is consistent with the inability to detect
infectious virus in the lungs of animals at the same stage
of the disease (Table 2). No antigen-positive cells were
seen in any of the tissues of animals which recovered
from the clinical disease, the majority of which were
examined at least 30 days after primary infection.
Recovery of virus and the detection of MHV-68 DNA
from the spleens of infected mice
A common biological feature of herpesviruses is their
ability to persist in the host and to reactivate periodically, resulting in the release of infectious virus. Gamma
herpesviruses characteristically establish a latent infection within lymphocytes. In an attempt to determine the
site of persistence of MHV-68, a variety of tissues were
co-cultivated with permissive cells to isolate latent/persistent virus. In three separate experiments, 3- to 4-weekold BALB/c mice were inoculated intranasally with 4 ×
105 p.f.u. MHV-68. In the first experiment, two mice
were killed on days 3, 5 and 10 post-infection, and cells
from the spleen, thymus, mesenteric lymph nodes and
blood were co-cultivated with BHK-21 cells. By using
this technique, virus was readily recovered from the
dissociated spleens of acutely infected animals after 5
days in culture (Table 3). These results contrast with
those obtained by direct homogenization and assay of
spleens, in which only low levels of infectious virus could
be detected at similar times post-infection (Table 1).
That the spleen is a site of persistent/latent virus was
confirmed in two additional experiments. In experiment
2, spleen cells from mice killed 36 days after infection
were found to carry persistent/latent virus.
In the third experiment, five mice were killed 90 days
post-infection, and the organs were removed immediately and divided into two equal parts. One part of each
organ was used for direct tissue homogenization and
assay for the presence of infectious virus, and the other
part for co-cultivation of lymphoid cells or tissue
explants to detect persistent/latent virus. Infectious virus
could not be detected in any of the splenic tissues assayed
by direct homogenization, whereas the spleens from
three of five mice assayed following dissociation of the
tissue and co-cultivation with permissive BHK-21 cells
led to the recovery of virus (Table 3), implicating this
organ as a major site of virus persistence. These data are
in accord with the detection of MHV-68 DNA in the
spleens of mice 30 days after infection (Fig. 6a). It is
noted that few positive cells are present in the germinal
centres of the spleen; this location tentatively suggests
that the infected cells may be of B lymphocyte origin.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Tue, 02 May 2017 18:34:59
MHV-68 infection of mice
Fig. 4. Histopathology of lung following infection with MHV-68.
Formalin- fixed, haematoxylin-stained sections of the lung at various
times after infection. (a) Lung of an uninfected mouse (3 to 4 weeks
old). A, Alveoli; B, bronchiole. (b) Lung 3 days post-infection with
MHV-68. B, Bronchiole; I, infiltration of mononuclear cells in
peribronchiolar and interstitial areas of the lung. (c) Lung of infected
mouse at 5 days post-infection. B, Bronchiole in which lumen contains
exudate of cellular debris; V, blood vessel (artery); I, an infiltrate of
mononuclear cells within the intima. (d) Lung of a clinically normal
infected mouse at 10 days post-infection. A, Alveoli; V, blood vessel
(artery); I, infiltration of mononuclear cells in perivascular and
interstitial areas. (e) Lung of a mouse 30 days post-infection. L,
Abnormal lymphoid accumulations can be observed in a subpleural
area. Similar lymphoid proliferations/accumulations could also be
observed in peribronchiolar and perivascular areas of the lung. Bar
marker represents 25 ~tm.
2353
Fig. 5. Immunoperoxidase staining of MHV-68 antigens in lung tissue
5 days after infection. Formalin-fixed lung sections were reacted with a
hyperimmune anti-MHV-68 serum and stained by the immunoperoxidase method. (a) Peribronchiolar and perivascular areas of the lung.
Arrows indicate that the peribronchial and perivascular cellular
infiltrate includes positively staining cells containing viral antigen. A,
Alveoli; B, bronchiole; V, blood vessel (vein). (b) Lung parenchyma.
Positive staining of enlarged alveolar epithelial cells can be seen (AE).
(c) A magnified view of the lung parenchyma shows positively stained
large mononuclear cells (MN) containing viral antigen. A, Alveolar
space. Bar markers represent (a) and (b) 20 ktm, (c) 8 ~tm.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Tue, 02 May 2017 18:34:59
2354
N. P. Sunil-Chandra and others
Fig. 6. D•t•cti•n•fMH•-68DNAinsp••enand•ung3•daysp•st-inf•cti•n.A•.2kbPstIr•p•atfragment•fMHV-68DNAwasused
to prepare a 3sS-labelled DNA probe for detection of latent viral DNA by in situ hybridization. (a) A few cells containing viral nucleic
acid could be detected within the lymphoid tissue of the spleen. The arrow indicates two positive cells located at the edge of a germinal
centre (G). The inset shows a magnified view which suggests that positive cells have just divided. (b) Positive cells were also seen in the
peribronchial tissue of the lung. Bar markers represent (a) 20 ~tm, inset 8 ~tm and (b) 40 ~tm.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Tue, 02 May 2017 18:34:59
MHV-68 infection of mice
Detection of viral DNA and the recovery of infectious
virus from the lungs of latently infected mice
Earlier studies on the pathogenesis of MHV-68 in both
newborn and 21-day-old outbred mice (Rajcani et al.,
1985, 1986) have identified the lung, trigeminal ganglia
and spleen as tissues in which virus can be detected by
direct homogenization and assay many days after
infection, thus implicating these organs as sites of
dynamic virus persistence. In our studies using inbred
BALB/c mice, virus could not be recovered either by
direct homogenization and assay or by co-cultivation of
tissue explants from non-lymphoid organs such as the
lung, heart, kidney, adrenal glands, brain and trigeminal
ganglia from five animals sampled 90 days postinfection. To increase the sensitivity of the assay
procedure, homogenization and assay of co-cultured
tissue explants was employed. This sensitive assay failed
to detect virus in explanted brain, trigeminal ganglia,
heart, kidney and adrenal glands removed from animals
90 days post-infection but virus was recovered from the
lungs of two of these five mice. This suggests that the
lung, in addition to the spleen, is a site of virus
persistence and is in agreement with the results of
Rajcani et al. (1985). Further evidence to support this
idea comes from a single observation of viral D N A
detection by in situ hybridization in the lungs of mice 30
days after infection (Fig. 6b). A small focus of positive
cells was observed, although these were not positive for
viral antigen by the immunohistochemical method.
Discussion
MHV-68 was originally isolated from free-living rodents
(bank vole) in Czechoslovakia and passaged in the brain
of newborn mice. In view of this neurological association
and the failure of the virus to react with sera specific for
murine cytomegalovirus, it was tentatively classified as a
member of the alphaherpesvirus subgroup (Svobodova et
al., 1982). However, analysis of the structure of the viral
genome and limited sequencing of viral genes have
revealed that MHV-68 is closely related to the gammaherpesviruses EBV and HVS (Efstathiou et al., 1990a, b).
As this is currently the only known gammaherpesvirus
naturally infecting mice, it provides an important
opportunity to study the properties of the virus in inbred
mice, and to compare and contrast these with the
properties of the primate gammaherpesviruses. In this
paper we have studied two routes of infection of BALB/c
mice with MHV-68, focusing on tissue tropism, pathological features of the infection and virus persistence.
Intranasal administration of MHV-68 to 3- to 4-weekold BALB/c mice resulted in the lung becoming the main
2355
tissue infected. Peak virus titres were detected 1 week
after infection, with viral antigens found in the alveolar
epithelium and a small number of mononuclear cells.
During this period sections of the lung revealed
peribronchiolar, perivascular and interstitial infiltration
of mononuclear cells among which there were foci of
necrosis and exudates containing cellular debris within
bronchioles. By day 10 post-infection, there was a
decrease in the amount of viral antigen detected, which
corresponds with the failure to isolate infectious virus
from the lung at this stage. These observations are
consistent with the findings of Rajcani et al. (1985), who
studied intranasal infection of 5-, 10- and 21-day-old
outbred mice. Following recovery from the primary
infection, MHV-68 DNA, but not viral antigen, was
detected in lung sections from some animals. In addition,
a pathological feature of the lungs at 30 days after
infection was the accumulation of lymphoid cells in the
sub-pleural and peribronchiolar regions, the architecture
of which resembled germinal centres. Lymphoproliferative responses of this type are common features of
gammaherpesvirus infections.
In addition to the lung, MHV-68 also infects the
kidney and heart, and to a lesser extent the spleen, during
the primary infection. However, we were unable to
isolate virus from the central or peripheral nervous
system, indicating that the nervous system is not a
primary target for infection, nor a site of virus
persistence. In contrast, the adrenal gland appears to be
heavily infected when the virus is introduced via the
bloodstream. This is a particularly interesting observation because both herpes simplex virus (an alphaherpesvirus) and murine cytomegalovirus (a betaherpesvirus)
are known to infect this tissue. The significance of virus
infecting the adrenal gland and the consequences for the
host are currently under investigation.
A major strategy employed by herpesviruses for
persisting in their host is the establishment of a latent
infection. In the sensory ganglia of mice infected with
herpes simplex virus, the presence of latent virus is
detected by co-cultivating the tissue with BHK-21 cells.
A similar approach was used to identify latent/persistent
MHV-68 in the spleen and lungs of BALB/c mice (Table
3). This observation, in conjunction with the detection of
the viral genome in the lung and spleen, suggests that
these tissues are the main sites of virus persistence. The
identification of latent virus in lymphoid cells by this
procedure is consistent with the properties of other
gammaherpesviruses, notably HVS and herpesvirus
sylvilagus (Falk et al., 1972; Rabson et al., 1971 ; Kramp
et al., 1985; Medveczy et al., 1984 ).
There are two pathological features of MHV-68
infection in mice that are similar to events in man
infected with EBV. Lymphoproliferation is observed in
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Tue, 02 May 2017 18:34:59
2356
N. P. Sunil-Chandra and others
the spleen of some animals and causes splenomegaly, a
condition that occurs in man during infectious mononucleosis. Furthermore, atrophy of the spleen and
thymus, seen in some BALB/c mice during the primary
infection, have also been observed in severe cases of
infectious mononucleosis. The cause of the latter is
unknown, but it is unlikely to arise from the cytopathic
effects of the virus because neither significant infectious
virus nor viral antigens are observed in these tissues.
From these initial studies on MHV-68 infection of
BALB/c mice, it would appear that certain features of
the primary and persistent infection resemble EBV
infection of man. We have obtained evidence (unpublished data), that the cell harbouring latent virus in the
spleen is a B lymphocyte. These observations suggest
that mouse gammaherpesvirus is likely to be a useful/
important animal model for understanding the detailed
virological and immunological processes in a natural,
permissive gammaherpesvirus infection.
The authors wish to thank the Medical Research Council of Great
Britain for supporting this work and A. C. Minson for helpful
discussions. N. P. S.-C. was supported by a grant from the Cambridge
Commonwealth Trust.
References
BLASKOVIC, D., STANCEKOVA, M., SVOBODOVA,J. & MISTRIKOVA, J.
(1980). Isolation of five strains of herpes viruses from two species of
free living rodents. Acta virologica 24, 468.
CIAMPOR, F., STANCEKOVA, M. 8/; BLASKOVlC, D. (1981). Electron
microscopy of rabbit embryo fibroblasts infected with herpesvirus
isolates from Clethrionomys glareolus and Apodemus flavicollis. Acta
virologica 25, 101 107.
EFSTATHIOU, S., HO, Y. M. & MINSON, A. C. (1990a). Cloning and
molecular characterization of the murine herpesvirus 68 genome.
Journal of General Virology 71, 1355-1364.
EFSTATHIOU, S., HO, Y. M., HALL, S., STYLES, C. J., SCOTT, S. D. &
GOMr'ELS,U. A. (1990 b). Murine herpesvirus 68 is genetically related
to the gammaherpesviruses Epstein-Barr virus and herpesvirus
saimiri. Journal of Gencral Virology 71, 1365-1372.
EPSTEIN, M. A., MORGAN, A. J., FINERTY, S., RANDLE, B. J. 8/;
KIRKWOOD, J. K. (1985). Protection of cotton top tamarins against
EB virus-induced malignant lymphoma by a prototype subunit
vaccine. Nature, London 318, 287-289.
FALK, L. A., WOLFE, L. G. & DEINHARDT, F. (1972). Isolation of
herpesvirus saimiri from blood of squirrel monkey (Saimiri sciureus).
Journal of the National Cancer Institute 48, 1499-1505.
FAZAKERLEY, J. K., SOUTHERN, P., BLOOM, F. & BOCI-IMEtER, M.
(1991). High resolution in situ hybridization to determine the cellular
distribution of lymphocytic choriomeningitis virus RNA in the
tissues of persistently infected mice: relevance to arenavirus disease
and mechanisms of viral persistence. Journal of General Virology 72,
1611-1625.
HONESS, R. W. (1984). Herpes simplex and 'the herpes complex':
diverse observations and a unifying hypothesis. Journal of General
Virology 65, 2077-2107.
HUNT, S. W. (1987). Preparation oflymphocytes and accessory cells. In
Lymphocytes: A Practical Approach. pp. 1-32. Edited by G. G. B.
Klaus. Oxford & Washington D.C:. IRL Press.
KRAMP, W. J., MEDVECZKY, P., MOLDER, C., HINZE, H. C. &
SULLIVAN, J. L. (1985). Herpesvirus sylvilagus infects both B and T
lymphocytes in vivo. Journal of Virology 56, 60455.
MEDVECZKY, P., KRAMP, W. J. & SULLIVAN, J. L. (1984). Circular
herpesvirus sylvilagus DNA in spleen cells of experimentally
infected cotton tail rabbits. Journal of Virology 52, 711-714.
MINSON, A. C. (1989). Herpesviridae. In Andrewes" Viruses of
Vertebrates, pp. 293-332. Edited by J. S. Porterfield. London:
Bailli6re-Tindall.
RABSON, A. S., O'CONNOR, G. T., LORENZ, D. E., KIRSCHSTEIN, R. L.,
LEGALLAIS, F. Y. & TRALKA, T. S. (1971). Lymphoid cell culture line
derived from lymph node of marmoset infected with herpesvirus
saimiri - preliminary report. Journal of the National Cancer Institute
46, 1099-1109.
RAJCANI, J., BLASKOVIC,O., SVOBODOVA,J., CIAMPOR, F., HUCKOVA,
D. 8£ STANEKOVA,D. (1985). Pathogenesis of acute and persistent
murine herpesvirus infection in mice. Acta virologica 29, 51~0.
RAJCANI, J., BUSTAMANTEDE CONTRERAS, L. R. & SVOBODOVA, J.
(1986). Corneal inoculation of murine herpesvirus in mice: the
absence of neural spread. Acta virologica 31, 25 30.
RICKINSON, A. B., YAO, Q. Y. 8/; WALLACE, L. E. (1985). Epstein-Barr
virus as a model of virus host interactions. British Medical Bulletin 41,
75-79.
RICKINSON, A. B., GREGORY, C. n., MURRAY, R. J., ULAETO, D. O. &
ROWE, M. (1989). Cell-mediated immunity to Epstein Barr virus
and the pathogenesis of virus-associated B cell lymphomas. In
Immune Responses, Virus Infections and Disease, pp. 59-83. Edited by
N. J. Dimmock & P. D. Minor. Oxford: IRL Press.
ROIZMAN, B. (1982). The family Herpesviridae; general description,
taxonomy, and classification. In The Herpesviruses, vol. 1, pp. 1-23.
Edited by B. Roizman. New York & London: Plenum Press.
SVOBODOVA, J., BLASKOVIC, D. & MISTRIKOVA, J. (1982). Growth
characteristics of herpes viruses isolated from free living small
rodents. Acta virologica 26, 256-263.
(Received 27 February 1992; Accepted 15 May 1992)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Tue, 02 May 2017 18:34:59