Download Murine gammaherpesvirus 68 establishes a

Journal o f General Virology (1992), 73, 3275-3279.
Printed in Great Britain
3275
Murine gammaherpesvirus 68 establishes a latent infection in mouse B
lymphocytes in vivo
N. P. Sunil-Chandra,* S. Efstathiou~ and A. A. Nash
Department o f Pathology, University o f Cambridge, Tennis Court Road, Cambridge C B2 1 Q P, U.K.
Murine gammaherpesvirus 68 (MHV-68) is able to
persist in spleen cells of infected mice. To determine
the cell type harbouring persistent virus, spleen cells
from infected animals were separated into immunoglobulin (Ig)-positive (B cell-enriched), Ig-negative (T cellenriched) and plastic-adherent (macrophage-enriched)
fractions. These cells were co-cultivated with permis-
sive BHK-21 cells in an infectious centre assay. The
consistent recovery and enrichment of infectious
centres in the Ig-positive fraction clearly demonstrates
that B cells are a major site of virus persistence/latency.
This observation indicates that MHV-68 is biologically
similar to Epstein-Barr virus and other members of the
B cell lymphotropic gammaherpesvirus 1 subgroup.
Gammaherpesviruses are generally considered to be
lymphotropic in nature, with the ability to establish
latent infections within lymphocytes (Roizman et al.,
1981; Honess, 1984). These viruses can induce a
lymphoproliferative disease in the infected host and can
efficiently immortalize lymphocytes infected in vitro, e.g.
Epstein-Barr virus (EBV) and herpesvirus saimiri
(HVS) (Shope et al., 1973; Crawford et al., 1982;
Fleckenstein & Desrosiers, 1982; Rickinson et al., 1989).
On the basis of the available data, gammaherpesviruses
have been subdivided into B cell-tropic (gammaherpesvirus 1), characterized by EBV and related viruses
of old world monkeys and apes, and T cell-tropic
(gammaherpesvirus 2), such as HVS and herpesvirus
ateles, both infecting new world monkeys (Honess,
1984). This classification may not hold true for all
gammaherpesviruses; for example, herpesvirus sylvilagus can establish a latent infection in both B and T cells
of cottontail rabbits (Kramp et al., 1985).
We have been studying murine herpesvirus 68 (MHV68), a naturally occurring murid herpesvirus originally
isolated from bank voles (Clethrionomys graleolus) in
Czechoslovakia (Blaskovic et al., 1980). Limited
sequence analysis of the MHV-68 genome has shown this
virus to be closely related to the gammaherpesviruses of
primates, EBV and HVS, in terms of both its gene
content and organization (Efstathiou et al., 1990a, b).
However, the overall genome structure and G + C
content of MHV-68 are most similar to those of the
gammaherpesvirus 2 group.
Studies on primary infection of BALB/c mice have
shown the lung to be the main tissue productively
infected by MHV-68, with virus present in alveolar
epithelium and mononuclear cells (Sunil-Chandra et al.,
1992). As with the other gammaherpesviruses, the spleen
appears to be the major site of virus persistence, with
latently infected cells detected by a co-cultivation assay.
This technique has been used widely to detect latently
infected lymphoid cells taken from animals infected with
HVS (Falk et al., 1972; Rabson et al., 1971) and
herpesvirus sylvilagus (Kramp et al., 1985; Medveczy et
al., 1984). The ability to recover virus by explant culture,
but not by direct homogenization of spleen, and the lack
of virus antigen expression at this site at late times postinfection (p.i.) (Sunil-Chandra et al., 1992), is taken by us
as a definition of virus latency.
The aim of the present study was to identify the
lymphocyte population harbouring latent MHV-68. To
achieve this, spleen cells were separated into plasticadherent cells, to enrich for macrophages, immunoglobulin (Ig)-positive cells (B cells) and Ig-negative ceils
(T cells) using anti-Ig-coated plates. This method offers a
rapid and specific means of separating lymphocyte
subpopulations (Nash, 1976; Mage et al., 1977; Mason et
al., 1987; Wysocki & Sato, 1978). The number of cells
harbouring latent virus from each subpopulation was
determined by an infectious centre assay (Sunil-Chandra
et al., 1992). In three separate experiments, 3- to 4-weekold BALB/c mice (Bantin and Kingman) were infected
intranasally with 4 x 105 p.f.u. MHV-68 (Sunil-Chandra
et al., 1992) and the spleen was removed on different days
p.i. Spleen cells were separated into plastic-adherent, Igpositive (B cells) and Ig-negative (T cells) fractions, as
shown in Fig. 1. Briefly, red blood cells (RBCs) were
t Present address: Division of Medical Virology, Institute of
Medical and Veterinary Science, Frome Road, Adelaide, SA 5000,
Australia.
0001-1189 © 1992 SGM
3276
Short communication
Spleen cell suspension
Water lysis of RBCs
IUncoated tissue culture platesI
Supernatant
37 °C 1 h
Adherent cells
............................
Sheep anti-mouse IgG-coated [
[ , tissue culture plates
I
Supernatant ~
:;:i:i;i;i:i:i:
~
~i~ifraction~i~;;;~
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~i~i:::::ii::i::!::ii::i::iiiiil
~ ...................
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~i!il):~:~i~i~i:~:~i~i~i~!ii~i:::i:!::::::ii~
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Fig. 1. Flow diagram to illustrate the process involved in separating
spleen lymphocytes using plastic dishes.
lysed by resuspending spleen cells in 1 ml disilled water
for 15 s and then in 10 ml of medium (RPMI 1640/20~
foetal calf serum). The cell suspension was washed once,
counted and used in the lymphocyte experiments.
Sheep anti-mouse I g G ( H + L ) afffinity-purified antibody (Sera Lab) was used to coat 60 mm tissue culture
grade plastic Petri dishes (Falcon) for the positive
selection of B cells by panning (Mage et al., 1977; Mason
et al., 1987). Briefly, 4 ml antibody solution (10 p.g/ml)
was added to a Petri dish for 16 to 18 h at 4 °C. The
antibody solution was then decanted and the dish was
washed with jets of PBS to remove any unbound
antibody. PBS (5 ml) containing 0.2~ BSA was added to
each plate and incubated for 30 min at room temperature
to block unbound sites on the dish. Excess albumin was
washed away and the dish was used for cell separation.
Plastic-adherent cells were separated by incubating
approximately 107 spleen cells in 5 ml medium in a
60 mm tissue culture grade Petri dish for 1 h at 37 °C in a
humidified 5 ~o COz incubator. Non-adherent cells were
resuspended and decanted. The cells present in 5 ml of
medium were added to a sheep anti-mouse IgG(H + L)coated Petri dish and incubated for 2 h at 4 °C. The nonadherent, Ig-negative cells were resuspended and decanted for use as the T cell-enriched fraction. Dishes
containing plastic-adherent cells and the Ig-positive cells
were washed three times to remove any unbound cells.
Finally, 2 x 106 BHK-21 cells in 5 ml medium were
added to the plastic-adherent and B cell-enriched dishes,
and also to the T cell-enriched fraction, and co-cultivated
at 37°C in a 5 ~ CO, incubator. After 5 days the
monolayers were fixed in 10~ formal saline, stained with
1~ toluidine blue and the number of infectious centres
was counted.
The results in Table 1 indicate clearly that MHV-68
could be recovered from the B cell-enriched fraction (Igpositive cells) and from the plastic-adherent cells early in
acute infection (i.e. days 5 and 7 p.i.). However, towards
the end of the acute disease and during the recovery
period (days 10 to 36), a high proportion of infectious
centres were recovered, predominantly from the B cellenriched fraction. The reduction in the number of
infectious centres observed at day 36 may indicate that
there is a loss of latent sites. This is supported by a
previous observation on the number of infectious centres
recovered at 90 days p.i. (Sunil-Chandra et al., 1992).
This suggests that, following the acute infection, the
immune system may regulate the number of latently
infected spleen cells.
In contrast, no or very few infectious centres were
detected in the T cell-enriched fraction. From these
studies it would appear that the principal cell harbouring
MHV-68 during the acute and latent infection is an Igpositive B lymphocyte. The mean number of infected
cells in the B cell population (Table 1) was found to be
approximately 1/105 spleen white cells, which is similar
to the number reported in patients with infectious
mononucleosis but more than that seen in B cells taken
from peripheral blood during a chronic EBV infection
(Rocchi et al., 1977).
To establish the percentage of macrophages, B and T
lymphocytes present in unseparated spleen cells, and
following separation on uncoated dishes or anti-Igcoated plates, flow cytometric analysis was carried out
using fluorescein isothiocyanate (FITC)-conjugated
monoclonal antibodies (MAbs) specific for B cells
(Ly5+), T cells (CD4 ÷ and CD8 ÷) and macrophages.
MAbs Ly5 (B220)-FITC and Macrph-FITC (Coulter
Immunology) were used at working dilutions of 1:20 in
PBS. Ly5 (B220) is expressed on pre-B cells, B cells and
some antibody-secreting cells including some plasma cell
tumours (Kincade, 1987). The antibody is from clone
RA36B2, rat IgG2b. MAb Macrph reacts with polypeptides specifically on macrophages and their precursors,
and is derived from clone M/70, rat IgG2a. Rat antimouse CD4 and CD8 MAbs labelled with FITC were
kindly provided by Dr S. Cobbold of this department,
and used as a mixture at working dilutions of 1 : 100 and
1:200, respectively, in PBS. Sheep anti-rabbit IgGFITC (Wellcome Diagnostics) was used as the negative
control antibody for direct immunofluorescence.
In Table 2, spleen cells were separated into different
populations as described, and stained with the various
FITC-labelled antibodies. The adherent cell fractions
were recovered for this purpose, either following
incubation in 5 mM-glucose solution for 15 min and
Short communication
T a b l e 1.
3277
Identification of spleen cell types harbouring virus during acute and latent MHV-68
infection
Time p.i.
(days)
Expt. 1
5
10
Expt. 2
20
Expt. 3
24
36
Total number
of spleen white
cells/spleen
( × 10-7)
B cell-enriched
population
T cell-enriched
population*
Plastic-adherent
cells t
2
3
2-5
4
1.1
12
18
0
0
0
0
58
4
36
4
352
288
282
1
0
1
0
0
18
13
0
25
32
0
26
10
16
Mouse
no.
1
1
4
2
3
1
2
3
2.6
2.8
2.7
2.3
1.4
Infectious centres/10~ spleen white cells
1
9
190
2
2
3
4
5
6
9
9
9
9
2.5
166
256
224
88
101
0
2
0
1
6
38
23
51
46
6
12
1
2
3
8
9.5
8
6.6
5.2
6-6
106
227
168
84
49
27
1
1
1
6
7
17
0
10
0
0
0
0
1
2
3
* Cells non-adherent to anti-IgG(H +L)-coated plates.
t Non-specifically adherent cells.
T a b l e 2. Flow cytometric analysis of spleen cell
subpopulations obtained using uncoated and anti-Ig-coated
tissue culture plates
Positive cells (~)*
MAb
Ly5 (B220)-FITC
Anti-CD4-FITC
and anti-CD8-FITC
Macrph-FITC
Total
spleen cells
B cellenriched
T cellenriched
Plasticadherent
48
49
74
26
3-5
88
57
24
6
6
6
14
* Ten-thousand cells were gated and the percentage of positive cells
was calculated from total cells gated in regions corresponding to
lymphocytes and monocytes.
pipetting (plastic-adherent cells), or incubating with 2 ~
mouse serum for 30 min and pipetting (Ig-positive cells).
Table 2 shows that 7 4 ~ of Ly5 + B cells were present in
the Ig-positive fraction compared to 3-5~ in the Ignegative T cell fraction. Failure to achieve a higher
percentage of B cells in the Ig-positive fraction is almost
certainly due to damage to cells following removal from
the plastic dishes (Mage et al., 1977; Wysocki & Sato,
1978). The detection of up to 57~ Ly5 + cells in the
plastic-adherent fraction helps to explain the presence of
infectious centres in these dishes. However, the number
of infectious centres recovered from these dishes was less
than would be expected from 57~ B cells, when
compared to the numbers recovered from 7 4 ~ B cells.
This suggests that a particular subset of B lymphocytes
may harbour the latent virus. The possibility that
macrophages harbour the virus in a latent form seems
unlikely because following their enrichment on plastic
dishes no concomitant increase in the number of
infective centres was observed.
To support the observation made above, we used
another technique for the separation of Ig-positive B
lymphocytes involving immunomagnetic beads (Kvalhelm et al., 1988, 1989). This method involves separating
spleen cells obtained from mice 20 days after an
intranasal infection. The spleen cells were coated with
rabbit anti-mouse I g G ( H + L ) and mixed with sheep
anti-rabbit IgG-coated magnetic beads (M-280 dynabeads; Dynal) according to the manufacturer's instructions. The numbers of cells in the magnetic bead-flee
fraction and the bead/cell rosette fraction were counted,
3278
Short communication
T a b l e 3. Recovery o f M H V - 6 8 from spleen B cells positively
selected 20 days p.i. b.v using immunomagnetic beads
Mouse
no.
Spleen cell
fraction
B cell-enriched;~
B cell-depleted
spleen cells
B cell-enriched
B cell-depleted
spleen cells
Ly5 + cells as
determined by flow
cytometry (%)*
Infectious
centres/107
cells~
77
11
106
7
74
10
36
5
* Ig-positive cells present in both B cell-enriched and -depleted
spleen cell fractions were stained with Ly5 (B220)-FITC MAb, which
binds Ly5 surface antigen. Ten-thousand cells were gated from each of
these cell preparations during flow cytometry. Percentage positive cells
was analysed in the region of lymphocytes and the cell/bead rosettes
(7000 B cell-enriched and 2000 B cell-depleted from each mouse).
t Number of infectious centres was determined from 107 cells of each
of these separated spleen cell fractions.
:~ Surface Ig-positive mouse spleen cells (B cells) were first allowed to
bind with rabbit anti-mouse IgG(H + L). This mouse B cell-rabbit antiIgG complex was then positively selected by using sheep anti-rabbit
IgG-coated magnetic beads (M-280 dynabeads).
cells were co-cultivated with BHK-21 cells to assay
infectious centres, and an aliquot from each fraction was
used for flow cytometry and fluorescence microscopy.
The results (Table 3) show that co-cultivation of the Igpositive fraction gives a greater number of infectious
centres than Ig-negative spleen cells. These results are
consistent with the relative percentages of Ly5+ cells in
both the Ig-positive cell-enriched and -depleted populations, as determined by fluorescence-activated cell
sorting (Table 3), and therefore supports the previous
observation that MHV-68 is indeed a B cell-tropic
gammaherpesvirus.
The data reported are consistent with murine B
lymphocytes, probably a subset, being the principal site
for virus persistence. In contrast to herpesvirus sylvilagus and members of the gammaherpesvirus 2 subgroup, mouse T lymphocytes were not a target for MHV68. This suggests that, biologically, MHV-68 is more
related to the gammaherpesvirus 1 subgroup, despite
having a genome structure more similar to that of the
gammaherpesvirus 2 subgroup. Further studies are
needed to identify the virus receptor and the nature of B
cells infected. In man, EBV enters B cells via CR2
(CD21) (Fingeroth et al., 1984). Although it is possible
that MHV-68 uses a similar receptor, other receptors
may also be involved because the virus infects a variety
of fibroblasts and epithelial cell lines (Svobodova et al.,
1982). Experiments are underway to identify the lineage
of B cells infected by the virus and the nature of the viral
genome during latency.
The authors wish to thank the Medical Research Council of Great
Britain for supporting this work, N.P.S.-C. was supported by a grant
from the Cambridge Commonwealth Trust.
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(Received 29 June 1992; Accepted 5 August 1992)