Download Presence of bovine viral diarrhoea virus in lymphoid cell populations

Journal of General Virology (1993), 74, 925 929. Printed in Great Britain
925
Presence of bovine viral diarrhoea virus in lymphoid cell populations of
persistently infected cattle
O. J. L o p e z , t F. A. O s o r i o , * C. L. Kelling and R. O. D o n i s
Department o f Veterinary Sciences, University o f Nebraska, Lincoln, Nebraska, 68583-0905, U.S.A.
Cattle infected in utero with bovine viral diarrhoea virus
(BVDV) often develop a lifelong persistent infection
(PI). During this PI, BVDV infects many cell types
including peripheral blood mononuclear cells
(PBMNC). To define the lymphoid cell populations in
which BVDV persists PBMNC subpopulations were
separated using monoclonal antibodies to cell surface
markers. Separated cells were analysed by a sensitive
PCR assay for BVDV, in conjunction with flow
cytometry to identify antigen-containing cells and with
viral infectivity assays. The results indicate that BVDV
establishes a productive PI in monocytes and T cells
bearing the marker BoCD4, BoCD8 or gamma-delta T
cell receptor. BVDV was not detected in B cells as a
productive nor a latent infection.
Bovine viral diarrhoea virus (BVDV) is one of the most
important pathogens of cattle. Its genome is a singlestranded (positive polarity) non-polyadenylated RNA
molecule of about 12.5 kb in length. BVDV and the other
members of the Pestivirus group, border disease virus
and hog cholera virus, have been classified as members of
the family Flaviviridae (Collett et al., 1989; Horzinek,
1991).
Field BVDV isolates can be divided into two biotypes
according to their cytopathic effect in cell cultures (Lee &
Gillespie, 1957; Gillespie et al., 1960). Infection with the
non-cytopathic biotype during early gestation leads to
the birth of persistently infected (PI) calves (McClurkin
et al., 1984). PI animals are immunologically tolerant to
the non-cytopathic BVDV strain which infects them
(McClurkin et al., 1984), but if superinfection with a
related cytopathic biotype takes place, or if a cytopathic
biotype arises from persisting BVDV, the animal will
usually die of mucosal disease (Bolin et al., 1985b;
Brownlie et al., 1984).
It has been demonstrated that calves infected with
BVDV exhibit an alteration of some immune functions
(Reggiardo & Kaeberle, 1981; Roth & Kaeberle, 1983;
Brownlie et al., 1984; Roth et al., 1986; Brown et al.,
1991) and this correlates with in vitro parameters (Atluru
et al., 1979, 1990). Moreover, a link has been found
between BVDV infection and enhancement of concurrent
infections (Edwards, 1986).
BVDV has affinity for cells of the immune system
either in acutely infected or PI animals (Bielefeldt
Ohmann et al., 1987; Bolin et al., 1987; Bolin & Ridpath,
1990). Acute infection of calves produces a decrease in
the number of leukocytes (Bolin et al., 1985 a; Ellis et al.,
1988), and BVDV antigen has been detected in PI cattle
in T, B and null lymphocytes, as well as in monocytes
(Bielefeldt Ohmann et al., 1987), platelets (Corapi et al.,
1989) and many other cell types (Allan et aI., 1989;
Bielefeldt Ohmann, 1983). Presence of virus was also
observed by electron microscopy of lymphoid cells
(Bielefeldt Ohmann et al., 1988).
The mechanism that leads to tolerance in PI with
BVDV and the kind of interaction that takes place
between the virus and the cells of the immune system are
unknown (Moennig, 1990). Increased knowledge of the
infected cell populations, and the functional alterations
caused by this infection, will help the understanding of
the degree of immunosuppression caused by the viral
infection in the acute, persistent and mucosal disease
phases of the disease. We report a study of the cellular
tropism of non-cytopathic BVDV in naturally PI cattle.
We analysed five PI animals from three different herds
for 8 months. The animals consisted of one 4.5 year old
cow, a 1"5 year old calf, and two heifers and one steer of
approximately 3 years of age. We focused on the
detection of BVDV in purified lymphoid populations
using techniques including detection of viral antigens by
flow cytometry, isolation of infectious virus and detection
of a conserved BVDV genomic sequence (Collett et al.,
1988) by reverse transcription of the viral RNA followed
by PCR (RT-PCR) (Lopez et al., 1991). As controls for
~Present address: Bio-Nebraska, 3940 Cornhusker, Suite 600,
Lincoln, Nebraska 68504, U.S.A.
0001-1201 © 1993 SGM
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Short communication
these experiments we used peripheral blood mononuclear
cells from two 9 month old calves from a beef herd raised
at the Agricultural Research and Development Center of
the University of Nebraska that is maintained free of
BVDV, bovine herpesvirus 1 and bovine leukaemia virus
infections. The control animals were maintained in
isolation and monitored (by serology and viral isolation)
for possible BVDV infection, throughout the whole
experiment.
F o r positive selection and characterization of lymphoid populations we used a panel of monoclonal
antibodies (MAbs) directed against the following specific
bovine cell (Bo) surface markers: I L R A D 11 against
BoCD4, I L R A D 15 against BoCD 1 l b (Mac 1), I L R A D
21 against monomorphic determinant class II (Mo),
I L R A D 29 against gamma-delta T cell receptor, I L R A D
30 against IgM (mu chain), I L R A D 42 against BoCD2
and I L R A D 51 againsty BoCD8. All of these MAbs
belong to a collection specific for bovine leukocyte
surface markers developed by the International Laboratory for Research in Animal Disease (ILRAD),
Nairobi, Kenya.
Blood was collected, after venipuncture from the
jugular vein, in tubes with 37.50 mg/ml EDTA. The
tubes were centrifuged at 800 g for 20 rain at 18 °C, and
the buffy coat was extracted and diluted in Ca2+/Mg ~+free PBS. To separate mononuclear cells from other cell
types, the buffy coat suspension was loaded onto
Lymphopaque (density 1"086 g/ml ; Nygaard and Co.),
and centrifuged at 800 g for 20 min at 18 °C. After three
washes with 1% albumin (fraction V ; Sigma) and 0.04 %
sodium azide in PBS, 25 gg of purified MAb against
the appropriate cell surface marker was added to 106
mononuclear cells and incubated for 45 min at 4 °C.
A goat anti-mouse IgG FITC conjugate (BoehringerMannheim) was used as second antibody. After staining,
cells were fixed in 3 % formaldehyde in PBS for 1 h and
washed four times in PBS. Cells were examined within
1 week for fluorescent emission in a Coulter 741 flow
cytometer.
In order to study the extent of BVDV infection in
peripheral lymphoid cells, different cell populations were
purified by panning (Lewis & Kamin, 1980), as follows.
Briefly, 5 x 107 total peripheral blood mononuclear cells
in 5 ml M E M plus 5 % fetal calf serum were loaded into
Petri dishes coated with the MAbs directed against the
appropriate cell surface markers as described previously,
and incubated for 1 h at 4 °C. After rinsing with PBS to
remove the floating cells, the attached cells were released
by gentle scraping and washed twice in M E M plus 5 %
fetal calf serum. The efficiency of purification was over
97 % in every case, as assessed by cell surface fluorescence. The presence of BVDV in the unfractionated
or separated mononuclear cell populations was assayed
by BVDV antigen detection, isolation of infectious
BVDV and detection of viral RNA.
In order to determine the proportion of productively
BVDV-infected lymphoid cell populations we carried out
semi-quantitative infectivity studies on positively selected
single-cell populations. We used bovine testicle (BT) cells
to detect the release of infectious BVDV from separated
lymphoid populations from three PI animals. The BT
cell line (established by R. O. Donis & E. J. Dubovi, New
York State Diagnostic Laboratory, Cornell University,
Ithaca, N.Y., U.S.A.) is free of BVDV and mycoplasma
infections.
Decreasing 10-fold dilutions (range 105 to 101) of
separated cells from PI animals were loaded onto four
32 mm well plates with BT cells. It was not necessary to
assay more than 105 cells because they were over 95 %
pure. After two consecutive passages, BT cells were
trypsinized and loaded onto slides coated with polylysine
(1 mg/ml) for 1 h at 4 °C and fixed for 10 rain in acetone,
at room temperature. An indirect fluorescence assay
using MAb 15C5 (see below) or bovine hyperimmune
serum, anti-BVDV (NADL), as first antibodies was
performed. In three PI animals from three different
herds, between 100 and 1000 purified monocytes or T
cells were enough for detection of BVDV. However, up
to 105 B cells were consistently negative by this procedure
(Table 1).
Table 1. Distribution of BVD V antigen, infectious
particles and BVDV RNA in purified peripheral blood
populations
Minimum number
of cells required for
BVDV detection
Cell marker
recognized by MAb
CD2+ (ILRAD 42)
IgM+ (1LRAD 30)
CD4+ (ILRAD 11)
CD8+ (ILRAD 51)
Mo (ILRAD 21)
Gamm~delta (ILRAD 29)
Total
Total (unrelated MAN[)
Antigen (%)*
Virust
PCR:~
PI 6.16_+2.25
C 2.23_+1.15
PI 0.72+0.40
C 0.84_+0'67
PI 6.77_+2.94
C 1.45_+0"76
PI 6.55__2.58
C 0.94-+0'36
PI 10.19+__4.39
C 1.04-+0.78
PI 5.46_+0"98
C 0.60_+0.16
PI 16-85±5-14
C 1.68_+0.66
PI 0.71_+0.25
C 0.66+0.66
103
103
-§
-§
l0s
103
103
103
103
103
103
103
10a
103
* Detection of BVD¥ antigen by flow cytometry. Mean values (%)
of fluorescentcells are given for PI and control (C) groups.
t Infectiousvirus.
:~Detection by PCR.
§Negative in 10~cells.
PlMAb against murine light (kappa) immunoglobulinchain.
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A panel of MAbs developed against the Singer strain
and specific for gp54 and gp48 (Donis et al., 1988;
Collett et al., 1989) was assayed for immunofluorescence
on BT cells infected with buffy coat and sera from the PI
animals. Out of 11 MAbs, only one (15C5) reacted with
the isolates from the three different herds. This MAb
reacted with gp48 of BVDV.
Total or purified single lymphoid cell populations were
fixed with 3 % formaldehyde in PBS for 1 h at room
temperature and were then washed and resuspended in
100 mM-ammonium chloride for 10 min at room temperature. They were permeabilized by incubation for
30 min in 0.05 % NP40 in PBS, washed four times in PBS
and incubated with MAb 15C5 for 45 min at 4 °C. After
two washes in PBS, the samples were incubated for
30 rain with FITC-conjugated anti-mouse IgG. After
three washes, the stained cells were assessed by flow
cytometric analysis within a week.
Viral antigens were detected in all peripheral mononuclear populations examined except in B cells (Table
1). The percentage of T cells containing BVDV antigen
was similar (no significant differences, P < 0'05, were
found) in total T cells and/or T4, T8, gamma~telta T cell
populations. However, a higher proportion of monocytes
(P < 0.05) were positive for viral antigen than the other
cell populations.
Our detection of BVDV in ILRAD 29 + cells (Table 1)
confirms the results of another laboratory (Bielefeldt
Ohmann et al., 1988). This population, originally
described as a null cell type, because of the lack of typical
B or T markers, is currently thought to carry the
gamma-delta T cell receptor. Cells carrying this receptor
constitute an important T cell subpopulation in the
bovine species (Hein & MacKay, 1991).
To detect BVDV RNA in lymphoid cells, we performed specific reverse transcription coupled with DNA
amplification of RNA obtained from different single cell
populations. Known aliquots of 101 to 105 were digested
for 10rain at room temperature with proteinase K
(200 gg/ml) and 20 min at 56 °C after the addition of
SDS (0.2% w/v). This solution was extracted with
phenol-chloroform and the RNA was precipitated with
cold 100 % ethanol and 3 N-sodium acetate pH 5.2 on
solid CO~ for 1 h. For the precipitation of viral RNA,
100 ng of DNA from uninfected BT cells was used as the
carrier. The pellet was dried in a desiccator and
resuspended in 10 gl of water. For PCR a pair of primers
was used that amplified a segment of 207 bp at the 3' end
of the BVDV genome (Lopez et al., 1991). Both primers
(20 gmol) were added to the resuspended pellet, heated
for 5 rain at 70 °C for RNA denaturation, and incubated
at 42 °C for 1 h after addition of 2 gl of Schimke solution
(comprising 50 mM-DTT, 50 mM-MgC12, 350 mN-KC1,
400 mN-Tris-HC1 pH 8), 2 ~tl of 2.5 mM-dNTPs (Phar-
927
macia), 0"5 gl of RNasin (Promega; 28 units/gl) and
1 gl of avian myeloblastosis virus reverse transcriptase
(Life Sciences; 19-6 units/ml). For the PCR assay 5 gl of
this solution was then used.
PCR was conducted as described previously (Lopez
et al., 1991). Briefly, 10 ~1 of 10 x amplification buffer,
16 gl of an equimolar mixture of dNTPs (5 mM), 3 gl of
each primer (40 lamol) and 5 gl of reverse transcription
solution were added, in a total volume of 100 gl. The
mixture was heated at 95 °C for 5 min, after which 3 units
of Taq polymerase (Promega) in a 5 gl volume of 1 x
buffer was added. To prevent evaporation of reagents,
100 gl of Nujon mineral oil (Perkin Elmer) was added.
The amplification reaction consisted of 40 equal cycles in
an automated thermal cycler (Perkin-Elmer Cetus). Each
cycle was composed of 1 min at 95 °C for denaturation,
1 min at 55 °C for primer annealing and 2 min at 72 °C
for primer extension. Several negative controls were
added for detection of possible DNA carry over. A 30 bp
probe was used for detection of specific amplified
products after blotting of the electrophoresed gel. The
oligonucleotide was end-labelled with 32p and polynucleotide kinase. Viral RNA was then assayed in
different purified single cell populations of the PI
animals. In purified populations of T cells and monocytes, 1000 peripheral mononuclear cells were sufficient
to detect the BVDV RNA, but not in the B cell fraction,
where 105 cells were consistently negative (Table 1).
To maximize the possibility of detection of minimal
amounts of BVDV, we repeated the RT-PCR after two
consecutive passages of B cells co-cultured with BT cells.
BVDV RNA was then found in RNA extracted from
the BT cells co-cultured with the B cell fraction (data
not shown). To determine whether this result was due
to infectious particles released upon productive cell
infection or instead to extracellular virus physically
adsorbed to B cells, we performed experiments in which
peripheral mononuclear cells from the PI animals were
treated with trypsin after purification and immediately
before initiation of the co-cultures, as described by Bolin
& Ridpath (1990). Total and purified lymphoid cells
were resuspended in MEM plus 1% trypsin and were
incubated for 30 min at 37 °C (Bolin & Ridpath, 1990).
MEM with 10 % FCS was added after incubation and
the suspension was centrifuged for 10 min at 800 g. The
viability of the cell suspension as determined by trypan
blue staining was greater than 80 %.
Viral RNA was detected in cells co-cultured with the
non-trypsin-treated B cells and in the non-B (T with
monocytes) cells (B-depleted). At the same time, viral
RNA was detected in the B-depleted fraction pretreated
with trypsin, but not in the trypsin-treated purified B
fraction.
In summary, we have detected BVDV antigen,
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infectious particles and BVDV RNA in T cell populations and monocytes of peripheral blood from BVDV
PI cattle, but only a small amount of BVDV can be
detected in association with the B cells of these animals.
This BVDV infectivity, which can be removed by
enzymatic digestion of the B cells, probably represents
BVDV adsorbed externally to the B cells instead of virus
produced endogenously by replication.
Our inability to detect BVDV infection in B cells of PI
animals contrasts with reports from other laboratories
(Bielefeldt Ohmann et al., 1988; Bolin et al., 1987). Some
authors have found infectious particles in purified B cells
(Bolin et al., 1987) although others have not (Bielefeldt
Ohmann et al., 1987). The contrasting results could be
based on the use, by different laboratories, of different
cell separation/purification techniques. Bolin et al.
(1987) purified B cells by positive selection using a
polyclonal serum against bovine IgG and then tested the
purified single-cell population for infectivity on indicator
cells. In contrast, Bielefeldt Ohmann et al. (1987)
attempted to isolate virus from B cells negatively selected
using a panel of MAbs, but subsequent co-cultivation
yielded no virus. Here, we purified B cells by positive
selection using an anti-mu chain MAb.
Our failure to detect viral antigen in B cells also
contrasts with a report (Bielefetdt Ohmann et at., 1987)
where total peripheral blood cells were centrifuged onto
slides and a double staining technique was used to detect
cell markers with specific MAbs and viral antigens
with a BVDV-specific polyclonal serum. It could be
argued that our failure to detect antigen in B cells could
be caused by a lower sensitivity of our anti-BVDV gp48
MAb, which recognizes a single structural protein (Donis
et al., 1988), in contrast to the polyclonal serum used by
Bielefeldt Ohmann et al. However, our results are
supported by our simultaneous failure to detect viral
RNA in the same population using the highly sensitive
PCR.
The difference between studies could also be related to
tropism differences between our isolates and those of
others. Our BVDV isolates are different from the Singer
strain when tested against a panel of MAbs (data not
shown). Finally, B cells may be differentially resistant to
BVDV, depending on the physiological status of the PI
animal, and/or the developmental stage at which the
fetal infection (leading to PI) took place.
Although further studies on BVDV genome transcription and expression in lymphoid cells would be
pertinent in order to ascertain the exact type of infection
established in these cells, our simultaneous detection of
viral antigen, infectious particles and viral genome in the
different populations suggests that in the PI cattle of our
study, the BVDV-lymphoid cell interaction results in
productive infection.
Whether B cells are always resistant to BVDV in vivo
or their permissiveness for BVDV is related to the
physiological status of the animal (or the viral strain
causing PI) deserves further investigation. The inability
of BVDV to infect B cells in naturally occurring PI cattle
seems to indicate that there are restrictions to the
generalized tropism of the virus. This merits study of the
mechanism of BVDV penetration into specific lymphoid
cells and investigation of B cell resistance. We do not
know, however, when the block takes place although the
failure to detect BVDV RNA in B cells suggests that B
cells block BVDV infection at the penetration stage or an
early stage of the infectious cycle, prior to viral protein
synthesis.
We thank Emiann Boldman for excellent technical assistance, and
Judith Galeota for proof-reading this manuscript. We also thank Dr
S.-S. Alex Chen for critical review of this manuscript. This research was
supported by funds provided by Special Research Grant (Animal
Health) USDA No. 9~34116-5366. The manuscript has been assigned
Journal Series No. 9869, Agricultural Research Division, University of
Nebraska.
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(Received 1 July 1992; Accepted 11 December 1992)
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