Demonstration of bovine viral diarrhoea virus in peripheral blood mononuclear cells of persistently infected, clinically normal cattle


Bielefeldt Ohmann, H.; Rønsholt, L.; Bloch, B.

Journal of General Virology 68: 1971-1982

1987


Perpheral blood mononuclear cells (PBL) from cattle known to be persistently viraemic with bovine viral diarrhoea virus (BVDV) following a foetal infection, were examined for the presence of viral antigens and cell associated infectious virus. Using immunocytochemical techniques, physical separations of PBL subsets and virus isolation techniques (directly and by cocultivation) it was found that infection occured in B and T lymphocytes, monocytes and a group of cells designated null cells for lack of more specific classification. The latter three groups also supported viral replication, as infectious virus could be isolated from enriched cell populations. BVDV-like particles in cytoplasmic vesicles of PBL subsets were detected by electron microscopy.

J.
gen.
Virol.
(1987),
68,
1971-1982.
Printed
in
Great
Britain
1971
Key
words:
BVD
virus/blood
mononuclear
cells/persistent
infection
Demonstration
of
Bovine Viral
Diarrhoea
Virus
in
Peripheral
Blood
Mononuclear
Cells
of
Persistently
Infected,
Clinically
Normal
Cattle
By
H.
BIELEFELDT
OHMANN,
14
1
L.
RONSHOLT
2
AND
B.
BLOCH'
'Department
of
Veterinary
Virology
and
Immunology,
The
Royal
Veterinary
and
Agricultural
University,
13
Bulowsvej,
DK-1870
Frederiksberg
C
and
2
The
State
Veterinary
Institute
for
Virus
Research,
Lindholm,
Kalvehave,
Denmark
(Accepted
23 March
1987)
SUMMARY
Peripheral
blood
mononuclear
cells
(PBL)
from
cattle
known
to
be
persistently
viraemic
with
bovine
viral
diarrhoea
virus
(BVDV)
following
a
foetal
infection,
were
examined
for
the
presence
of
viral
antigens
and
cell-associated
infectious
virus.
Using
immunocytochemical
techniques,
physical
separations
of
PBL
subsets
and
virus
isolation
techniques
(directly
and
by
cocultivation)
it
was
found
that
infection
occurred
in
B
and
T
lymphocytes,
monocytes,
and
a
group
of
cells
designated
null
cells
for
lack
of
more
specific
classification.
The
latter
three
groups
also
supported
viral
replication,
as
infectious
virus
could
be
isolated
from
enriched
cell
populations.
BVDV-like
particles
in
cytoplasmic
vesicles
of
PBL
subsets
were
detected
by
electron
microscopy.
INTRODUCTION
Bovine
viral
diarrhoea
virus
(BVDV)
is
the
aetiological
agent
of
mucosal
disease
(MD)
in
cattle,
and
may
cause
a
whole
range
of
other
clinical
conditions,
now
included
in
the
BVD
syndrome
(Stober,
1984;
Bielefeldt
Ohmann
&
Babiuk,
1986
b).
BVDV
is
currently
classified
as
a
member
of
the
Pestivirus
genus
of
the
non-arbo
togaviruses
(Westaway
et
al.,
1985).
The
virus
has
a
marked
tropism
for
lymphoid
and
epithelial
(keratinized
and
mucosal)
tissues
(Bielefeldt
Ohmann,
1983)
and
in
vitro
it
can
replicate
in
cell
cultures
derived
from
such
tissues.
For
decades
the
pathogenesis
of
MD
remained
a
complete
enigma.
Animals
succumbing
to
MD,
which
is
an
inevitably
fatal
course
of
the
infection,
are
viraemic
and
virus
can
be
isolated
from
almost
every
tissue.
However,
whether
the
course
of
the
disease
is
acute
or
more
protracted,
perhaps
even
chronic,
extending
over
several
months,
the
animals
usually
fail
to
develop
BVDV-specific
neutralizing
antibodies.
This
may
suggest
some
kind
of
virus-specific
tolerance.
Very
recently
some
understanding
of
the
mechanisms
involved
in
the
disease
have
emerged.
Thus,
it
has
been
shown
that
MD
only
develops
in
cattle
persistently
infected
with
non-cytopathic
BVDV,
established
by
foetal
infection
during
the
first
3
months
of
gestation
(McClurkin
et
al.,
1984;
Roeder
&
Drew,
1984;
Brownlie
et
al.,
1984).
This
early
infection
induces
an
apparent
tolerance
to
the
virus
and
the
virus
persists
and
continues
to
replicate.
The
tolerance
appears
to
be
virus
strain-specific,
but
whether
it
is
absolute
and
invariably
permanent
seems
controversial
(Gardiner
et
al.,
1983;
Terpstra,
1981;
Westbury
et
al.,
1979;
L.
Ronsholt,
unpublished
data).
The
question
that
is
central
to
the
understanding
of
the
pathogenesis
of
MD,
i.e.
exactly
what
precipitates
the
disease
in
apparently
healthy,
persistently
infected
animals,
has
also
created
much
controversy.
Various
hypotheses
for
these
precipitating
factor(s)
have
been
put
forward
such
as
superinfection
with
heterologous
BVDV
strains
(Liess
et
al.,
1983;
Brownlie
et
al.,
1984;
Bolin
et
al.,
1985),
the
emergence
of
a
cytopathic
mutant
of
the
persisting
virus
strain
(Brownlie
et
al.,
1986),
hormonal
changes
related
to
puberty
(Roeder
&
Drew,
1984)
or
superinfection
with
an
agent
unrelated
to
BVDV
(Littlejohns
&
Walker,
1985).
t
Present
address:
Veterinary
Infectious
Disease
Organization,
124
Veterinary
Road,
Saskatoon,
Saskatchewan,
Canada
S7N
OWO.
0000-7559
©
1987
SGM
1972
H.
BIELEFELDT
OHMANN,
L.
RONSHOLT
AND
B.
BLOCH
Questions
pertinent
to
the
understanding
of
the
pathogenesis
concern
the
mechanism(s)
governing
the
establishment
and
maintenance
of
the
persistent
virus
infection,
and
the
identity
of
the
cell(s)
harbouring
and
replicating
the
virus.
Virus
can
be
isolated
from
buffy
coat
cells
of
the
peripheral
blood
and,
being
circulating
cells,
these
may
serve
as
vehicles
for
the
dissemination
of virus
to
other
target
organs.
The
aim
of
the
present
investigations
has
been
to
identify
the
cell
type(s)
in
the
peripheral
blood
of
persistently
infected,
clinically
healthy
cattle
that
harbours
and
perhaps
replicates
the
virus.
METHODS
Animals.
Eleven
calves
(four
non-infected
and
seven
persistently
infected
following
an
experimental
infection
with
a
mixture
of
cytopathic
and
non-cytopathic
BVDV
strains
in
the
first
3
months
of
foetal
life)
were
bled
regularly
over
a
9-month
period
(age
10
to
19
months).
Two
of
the
persistently
infected
calves
succumbed
to
clinical
MD
during
the
investigation
period.
The
remaining
nine
(four
non-infected
and
five
infected)
were
healthy
at
the
age
of
18
to
20
months.
A
separate,
persistently
viraemic
blood
donor
calf
was
the
offspring
of
a
cow
inseminated
with
a
BVDV-containing
semen
sample
(A.
Meyling,
unpublished
data).
Blood
samples
were
also
obtained
from
two
clinically
healthy
yearlings,
naturally
infected
(viraemic)
with
BVDV
and
identified
as
such
during
a
control
examination.
Additional
blood
samples
were
drawn
from
clinically
healthy,
seropositive
cattle
of
all
ages
and
different
breeds.
Peripheral
blood
mononuclear
cell
preparations.
Blood
was
drawn
by
venipuncture
either
into
syringes
containing
citrate—dextrose
or
into
syringes
and
defibrinated
mechanically
with
glass
beads;
the
peripheral
blood
mononuclear
cells
(PBL)
were
isolated
by
density
centrifugation
on
Ficoll-Hypaque
(Pharmacia)
as
previously
described
(Bielefeldt
Ohmann
et
al.,
1983).
Adherent
(AD)
cells
were
depleted
by
incubating
5
x
10
7
to
10
x
10'
cells,
suspended
in
5
ml
RPMI
1640
(Gibco)
with
10%
horse
serum
(HS;
Gibco)
in
25
cm
2
tissue
culture
flasks
(Nunc)
which,
prior
to
cell
seeding,
had
been
coated
by
incubation
for
1
h
at
38
°C
with
heat-inactivated
foetal
bovine
serum
(found
to
be
devoid
of
BVDV-specific
antibody
and
BVDV).
After
incubation
for
2
h
at
38
°C,
the
non-adherent
(NA)
cells,
resuspended
in
the
medium,
were
collected
and
pooled
with
the
first
aliquot
of
wash
fluid
(RPMI).
Further
depletion
of
monocytes
(as
well
as
some
B
cells,
see
Table
5)
from
the
plastic-NA
cells
was
accomplished
by
Sephadex
G-10
column
passage
as
previously
described
(Bielefeldt
Ohmann
et
al.,
1983)
except
that
nylon
wool
was
used
in
place
of
glass
beads.
The
plastic-AD
cells
were
washed
several
times
in
cold
Ca
2
+-
and
Mg
2
+-free
phosphate-buffered
saline
(PBS)
and
incubated
for
5
min
with
ice-cold
EDTA.
Final
detachment
of
the
cells
was
accomplished
by
vigorous
pipetting.
They
were
then
washed
twice
in
cold
PBS,
counted
and
employed
in
the
various
assays.
The
viability
of
the
AD
cells
was
always
more
than
98%,
as
indicated
by
trypan
blue
exclusion.
Depletion
of
subpopulations
by
treatment
with
monoclonal
antibody
(MAb)
and
complement.
Complement
(C')-
mediated
lysis
of
PBL
subpopulations,
defined
by
reactivity
with
MAb
(see
below
and
Table
1)
was
accomplished
as
described
previously
(Bielefeldt
Ohmann
et
al.,
1985).
The
complement
source
was
rabbit
serum
(Glapco
Aps,
Aarhus,
Denmark
or
Cedarlane
Laboratories,
Hornby,
Ont.,
Canada).
After
determining
the
proportion
of
dead
cells
by
staining
with
trypan
blue,
the
dead
cells
were
removed
by
differential
centrifugation
on
Ficoll-Hypaque.
Treatment
with
L-leucine
methyl
ester
(L-LME).
PBL
were
treated
with
L-LME
(Sigma)
in
order
to
deplete
the
monocytes,
following
a
protocol
adapted
for
the
bovine
system
(Bielefeldt
Ohmann
et
al.,
1985).
The
L-LME
was
used
at
a
final
concentration
of
10
mm.
In
some
experiments
an
attempt
was
made
to
remove
damaged
and
dead
cells
by
centrifugation
on
Ficoll-Hypaque.
The
purity
of
the
cell
populations
obtained
by
the
above
methods
are
given
in
Table
5.
Cell
yields
varied
with
the
method
employed
(see
cited
literature).
MAbs.
Murine
MAbs
with
specificity
for
bovine
leukocyte
populations
were
obtained
from
Dr
W.
C.
Davis
(Washington
State
University,
Pullman,
Wash.,
U.S.A.)
and
Dr
A.
J.
Teale
(ILRAD,
Nairobi,
Kenya).
Table
1
summarizes
the
reported
properties
of
these
MAbs.
CH128A,
CH61A
and
B26A(4)
detect
the
same
cell
population.
However,
B26A(4)
shows
poor
reactivity
on
fixed
cells,
but
binds
complement
well.
For
similar
reasons
Plg45A,
reactive
with
bovine
B
cells,
was
only
employed
for
the
antibody-complement
lysing
procedure
(see
above).
Three
MAbs,
TH14B,
TH81A
and
H42A,
react
with
bovine
major
histocompatibility
complex
(MHC)
class
II
antigens
(Ia-like
antigens)
which
are
the
equivalents
of
human
DR,
DQ
and
DP,
respectively.
These
antigens
are
present
on
B
lymphocytes
and
on
most
monocytes.
Histochemistry
and
immunocytochemistry
(ICC).
Cytospins
(cytospin
centrifuge;
Shandon
Southern
Products
Ltd.,
Runcorn,
U.K.)
were
made
on
cleaned
poly-L-lysine
(mol.
wt.
500K;
Sigma)-coated
glass
slides,
from
all
cell
preparations.
One
set
was
fixed
in
formaldehyde
and
stained
for
non-specific
a-naphthyl
butyrate
esterase
(Koski
et
al.,
1976),
in
order
to
detect
cells
of
the
mononuclear
phagocyte
lineage
(Bielefeldt
Ohmann
et
al.,
1983).
Another
set
of
cytospin
preparations
for
ICC
were
fixed
in
acetone—chloroform
(1
:1)
for
8
min
with
thorough
air
drying
both
before
and
after
fixation.
The
slides
were
stored
at
—20
°C
in
sealed
boxes
until
staining
could
be
performed.
Designation
BVDV
in
blood
of
persistently
infected
cattle
Table
1.
Reactivity
of
MAbs
1973
of
MAb
Isotype
Reactivity
Source/reference
BIg73A
IgG,
Bovine
IgM,
B
cells
W.
C.
Davis
(personal
communication)
PIg45A
IgG
2B
IgM
Davis
et
al.
(1987)
CH128A
IgG,
All
T
cells
(E
rosette
receptor)
Davis
et
al.
(1987)
CH61A
IgG,
All
T
cells
Davis
et
al.
(1987)
B26A
IgM
All
T
cells
Davis
et
al.
(1987)
DH59B
IgG,
Monocytes,
granulocytes
Davis
et
al.
(1987)
TH14B
IgG2A
MHC-II
(equivalent
to
human
DR)
Davis
et
al.
(1987)
TH8
IA
IgG2A
MHC-H
(equivalent
to
human
DQ)
Davis
et
al.
(1987)
H42A
IgG
2A
MHC-II
(equivalent
to
human
DP)
Davis
et
al.
(1987)
IL-Al2
IgG2A
BoT4,
30%*
Baldwin
et
al.
(1986)
Morrison
et
al.
(1986)
IL-A17
IgG,
BoT8,
20%*
Morrison
et
al.
(1986)
IL-A23
IgG,
Macrophage
differentiation
antigen;
1-11%*
Ellis
et
al.
(1986)
*Percentages
mentioned
are
the
proportion
of
peripheral
blood
leukocytes
in
normal,
adult
cattle
positive
for
the
antigen,
as
determined
by
fluorescence-activated
cell
sorting
analysis.
The
protocols
for
single
and
double
immunolabelling
for
detection
of
either
virus
or
leukocyte
antigen
or
both,
respectively,
were
as
described
in
detail
elsewhere
(Bielefeldt
Ohmann,
1987).
Briefly,
BVDV
antigens
were
detected
by
indirect
immunoperoxidase
staining
of
cytospin
preparations
following
blocking
of
endogenous
peroxidase
activity
and
non-specific
antibody
binding.
The
primary
antibody
was
purified
swine
anti-BVDV
immunoglobulin
(Ig)G
and
the
conjugate
was
rabbit
anti-swine
Ig-horseradish
peroxidase
(RAS-HRP,
Dakopatts,
Glostrup,
Denmark).
The
specific
binding
was
visualized
with
a
0.05%
diaminobenzidine
(DAB)
substrate.
On
some
occasions
a
fluorescein
isothiocyanate-conjugated
rabbit
anti-swine
Ig
(Dakopatts)
was
used
for
visualization
of
infected
cells.
A
triple-step
alkaline
phosphatase-anti-alkaline
phosphatase
(APAAP)
system
was
used
for
leukocyte
antigen
detection.
Initially,
slides
were
incubated
with
the
appropriate
dilution
of
MAb
(Table
1),
followed
by
incubation
with
rabbit
anti-mouse
immunoglobulin
(Dakopatts)
and
finally
with
the
APAAP
complex
(Dakopatts,
code
no.
D
651).
Washing
sequences
of
4
x
5
min
in
PBS
were
performed
after
each
incubation.
The
reaction
was
visualized
by
incubating
the
slides
with
a
chromogen
substrate
(2
mg
naphthol
AS-MX
phosphate
in
04
M-Tris-
buffer
pH
82,
1
mM-levamisole
and
10
mg
fast
red
TR
salt).
After
counterstaining
with
methyl
green
the
cell
preparations
were
mounted
in
aqueous
gelatine
(Glycergel,
Dakopatts).
Double
immunolabelling
for
both
virus
and
leukocyte
antigen
was
performed
by
applying
the
sequences
mentioned
for
the
APAAP
system
and
including
swine
anti-BVDV
IgG
in
the
first
and
second
step
and
RAS-
HRP
in
the
third
step
(Bielefeldt
Ohmann,
1987).
For
visualization
of
the
two
enzyme
conjugates
the
slides
were
first
incubated
with
the
DAB
substrate
followed
by
washing
in
PBS
and
then
incubation
with
the
naphthol-fast
red
substrate.
Specificity
controls
for
BVDV
detection
consisted
of
adsorption
of
the
antiserum
to
BVDV-infected
monolayer
cultures,
fixed
in
100%
acetone
for
10
min.
As
controls
for
both
the
virus
and
the
leukocyte
antigens
were
also
used,
omission
of
either
of
the
antibodies,
replacement
of
the
primary
Ab
with
a
nonsense
Ab
(swine
anti-rotavirus
serum
in
the
case
of
BVDV,
MAbs
to
human
T4
or
T8
antigens
in
the
case
of
the
leukocyte
antigens)
or
with
normal
serum
from
the
species
of
origin.
Negative
controls
comprised
cell
preparations
from
BVDV-free
animals.
Two
or
more
slides
were
stained
for
each
antigen.
The
preparations
were
read
blind,
and
a
minimum
of
300
cells
per
slide
were
recorded.
Virus
isolation
and
titration.
Madin-Darby
bovine
kidney
(MDBK)
cells,
free
from
BVDV
and
mycoptasma
(kindly
provided
by
Dr
A.
Meyling)
were
grown
in
Eagle's
minimum
essential
medium
(MEM)
supplemented
with
L-glutamine
(292
mg/1,
Gibco),
sodium
bicarbonate,
gentamicin
(50
µg/ml,
Gibco),
and
HS
(7%
for
outgrowth
and
2%
for
maintenance).
Samples
of
the
various
cell
preparations
were
disintegrated
by
three
cycles
of
freezing
and
thawing
and
the
TCID"
of
lysed
material,
blood
plasma
(procured
after
the
initial
centrifugation
of
blood
for
PBL
isolation)
or
wash
fluid
from
the
PBL
isolations
was
estimated
by
twofold
titration
in
a
microtitre
system
(Nunc)
(Bielefeldt
Ohmann,
1981).
Using
three
or
four
wells
per
sample
dilution,
a
suspension
of
MDBK
cells
(1
x
10
5
cells
per
well)
was
incubated
with
the
inoculum
in
a
total
volume
of
100
ul
per
well
for
1
to
2
h
at
38
°
C.
Following
addition
of
100111
MEM
supplemented
with
8%
HS
to
each
well,
incubation
was
continued
for
72
h.
At
this
time
the
cultures
were
checked
for
c.p.e.
and
then
terminated.
Following
a
brief
wash
in
PBS,
the
cultures
were
dried
(1
h,
38
°C),
fixed
in
20%
acetone
in
0.85%
NaCI
with
0.02%
bovine
serum
albumin,
again
1974
H.
BIELEFELDT
OHMANN,
L.
RONSHOLT
AND
B.
BLOCH
Table
2.
Frequency
of
virus
antigen-positive
cells
among
PBL
and
yield
of
infectious
virus
Animal
no.
Virus-positive
cells*
(%)
TCID
50
/10
6
cellst
n1
6
18.3
+
3.5
32-512
8
KIO
16.3
+
1.5
160-512
4
TIO
13.6
+
2.3
128-254
5
13
9.3
+
1.4
64-188
4
14
30.3
+
3.3
128-256
7
144
24.4
+
3.4
128-1024
9
*
Determined
by
ICC
(see
Methods),
percentage
of
non-fractionated
PBL
(mean
±
s.D.).
t
Direct
isolation
from
lysates
of
PBL.
1
Number
of
independent
samplings
during
an
8
to
9
month
period.
dried
for
2
h
at
38
°C,
and
then
either
stained
immediately
for
BVDV
antigen
or
stored
in
sealed
bags
at
-
20
°C
to
be
stained
later.
For
immunocytochemical
detection
of
BVDV
antigen,
cultures
were
incubated
with
swine
anti-BVDV
IgG
for
20
min
at
38
°C,
followed
by
a
15
min
incubation
with
RAS-HRP.
The
plates
were
washed
five
times
in
PBS
with
1%
Tween
80
after
each
antibody
incubation.
The
immunolabelling
was
visualized
by
incubating
with
substrate
containing
2
mg
3-amino-9-ethylcarbazole
per
5
ml
0
1
m-acetic
acid/acetate
buffer
pH
5,
and
0.015%
H,0
2
.
Evaluation
of
the
labelling
was
either
performed
immediately
at
the
end
of
a
20
to
25
min
incubation
period,
or
substrate
was
replaced
by
a
4%
formaldehyde
solution
and
the
plates
were
stored
at
4
°C
for
reading
later.
Most
assays
were
performed
twice.
Virus
detection
by
cocultivation.
Since
subpassage
of
cocultures
of
leukocytes
and
MDBK
cells
did
not
increase
the
detectability
of
virus-replicating
cells
(data
not
shown),
we
turned
to
the
use
of
a
microsystem.
The
leukocyte
samples
were
seeded
in
quadruplicate
sets
of
two-fold
dilutions
in
96
well
tissue
culture
plates,
from
1
x
10
4
cells
per
well
to
several
dilutions
beyond
one
cell
per
well.
MDBK
cells
were
then
added
and
the
cocultures,
in
a
final
volume
of
200µl
of
RPMI
:MEM/5%
HS,
were
incubated
in
a
5%
CO
2
atmosphere
at
39
°
C
for
96
h.
At
this
time
the
cultures
were
checked
visually
for
c.p.e.,
fixed
and
stained
for
virus
antigen
as
described
above.
Electron
microscopy.
Pellets
of
the
various
cell
preparations
were
prepared
for
transmission
electron
microscopy
as
previously
described
(Bielefeldt
Ohmann
&
Bloch,
1982).
In
some
cases
the
leukocytes
were
labelled
in
the
living
state
for
surface
antigens
by
an
immunogold
technique
employing
the
MAbs
listed
in
Table
I
and
subsequently
prepared
for
electron
microscopical
examination
(Bielefeldt
Ohmann
&
Bloch,
1982;
H.
Bielefeldt
Ohmann,
B.
Bloch,
W.
C.
Davis
&
J.
Askaa,
unpublished
results).
Statistical
analysis.
Where
appropriate,
comparison
of
data
from
non-infected
and
infected
animals
was
done
using
Student's
t-analysis.
RESULTS
Non-cytopathic
BVDV
was
readily
isolated
from
serum,
plasma
or
cell
lysates
(after
freezing
and
thawing)
of
calves
with
known
foetal
exposure
to
the
virus
and
lacking
virus-specific
neutralizing
antibodies
at
the
age
of
6
months
and
onwards.
The
cell-related
virus
was
not
'carry-
over'
from
the
plasma
as
indicated
by
testing
the
third
washing
solution
following
Ficoll-
Hypaque
isolation
of
the
PBL.
This
solution
was
always
negative
for
virus
even
after
four
passages
(data
now
shown).
The
results
of
the
direct
virus
isolation
from
PBL
were
supported
by
immunocytochemical
detection
of
intracellularly
located
viral
antigen
in
5
to
36%
of
the
PBL.
Table
2
summarizes
the
results
obtained
from
six
calves
(omitting
the
data
from
a
large
number
of
repeatedly
virus-negative
animals).
Whereas
the
frequency
of
antigen-positive
cells
appeared
to
be
fairly
constant
for
a
particular
animal
during
the
investigation
period
(8
to
9
months),
differences
between
the
individual
calves
were
evident
(Table
2).
The
amount
of
infectious
virus
rescued
from
cell
lysates
of
PBL
varied
both
for
the
individual
animal
between
samplings
(ranges
shown
in
Table
2)
and
between
animals,
and
could
not
be
directly
related
to
the
frequency
of
infected
cells
(Table
2).
There
was
no
direct
correlation
between
the
frequency
of
virus
antigen-positive
cells
as
detected
by
cocultivation
of
PBL
with
MDBK
cells
(data
not
shown).
At
no
time
was
cytopathic
virus
isolated
from
any
of
the
calves
and
no
reversion
to
cytopathogenicity
was
seen
with
any
of
the
isolates
during
multiple
cell
culture
passages
(more
than
12).
BVDV
in
blood
of
persistently
infected
cattle
1975
Table
3.
Distribution
of
virus-infected
cells
among
PBL
subpopulations
in
persistently
infected
calves
Phenotypic
marker
(PM)
(monoclonal
antibody)*
Positive
cells
(%)t
A
III
PM
-
/V
-
PM+/V
-
PM
-
/VP
PM+/V+
B
cell
(IgM)
63.0
20.1
16.0
0.91
16
(BIg73A)
(49.0-78-5)
(12.0-29-0)
(4-0-29.0)
(0.0-4-5)
All
T
cells
49.4
30.1
12.2
8-3
7
(CH128/CH61A)
(42-0-56.7)
(21.0-41-7)
(8.7-17-0)
(2.7-14-0)
BoT8
63.4
16.8
14.6
5.1
10
(IL-A17)
(57-0-80.5)
(8.5-26.5)
(6-5-25.5)
(2.0-9-0)
Monocytes
68.4
12.7
14.8
4.1
13
(DH59A)
(57.5-81-0)
(6.5-18-5)
(3.5-26.5)
(0-5-14.0)
Monocyte
subpopulation
80.9
3.6
12.5
3.0
7
(Chl6A/CH137)
(65.5-94-5)
(0.5-7.0)
(4.0-22-0)
(0-0-6.5)
MHC-II
(DR
equivalent)
58.2
24.7
11.6
5.5
11
(TH14B)
(43.0-69.8)
(15.1-31-0)
(3.5-22-0)
(1-0-11.0)
MHC-II
(DQ
equivalent)
53.4
22.5
10.9
3.2
10
(TH81A)
(57-0-70.5)
(15.5-28-5)
(9.0-22-0)
(0.5-5-5)
MHC-II
(DP
equivalent)
59.0
22.8
13.3
4.9
9
(H42A)
(50.0-73-0)
(16.0-29.6)
(7.8-19.5)
(2.7-8-5)
*
The
codes
in
parentheses
refer
to
the
MAbs
used
for
the
detection
of
phenotypic
markers,
as
listed
in
Table
1.
t
Percentage
of
non-fractionated
PBL.
The
total
frequency
of
virus-positive
cells
(i.e.
PM
-
/V+
plus
PM
,-
/V+)
is
not
exactly
the
same
in
all
sets
because
these
were
not
generated
from
the
same
calves
in
all
cases.
The
averages
given
should,
therefore,
only
be
taken
as
indicative.
I
Number
of
animals
examined
(includes
repeated
examinations
of
some
animals).
Identification
of
virus-infected
cells
by
immunocvtochemical
procedures
Double
immunolabelling
procedures,
employing
murine
MAbs
against
bovine
leukocyte
antigens
(Table
1)
and
a
polyclonal
swine
IgG
against
BVDV
using
two
enzyme-conjugated
secondary
antibodies,
revealed
that
infected
cells
could
be
found
within
all
four
major
subgroups
of
cells
in
PBL,
i.e.
monocytes,
T
and
B
lymphocytes
and
null
cells
(Table
3
and
Fig.
1).
However,
each
subgroup
did
not
contribute
equally
to
the
infected
pool.
Based
on
data
included
in
Tables
2
and
3
it
can
be
estimated
that
T
cells
(CH128A+/CH61A+
cells)
constituted
40
to
50%,
B
cells
(BIg73A+/MHC-II+)
less
than
4%,
monocytes
(NSE+,
DH59B+/MHC-II+)
17
to
24%
and
null
cells
(non-T,
non-B,
non-macrophage
cells)
24
to
40%
of
the
virus
antigen-positive
cells.
The
persistent
infection
did
not
cause
any
significant
changes
in
the
total
leukocyte
numbers
in
the
blood
(not
shown),
but
shifts
in
the
frequencies
of
the
mononuclear
cell
subpopulations
were
apparent
(Table
4).
Thus,
the
frequencies
of
B
lymphocytes
(BIg73A+
cells)
and
monocytes
(DH59B+,
NSE+
cells)
were
elevated,
the
latter
significantly
(P
<
0.05),
while
the
frequency
of
T
lymphocytes
was
significantly
lowered
(P<
0.05)
in
the
persistently
infected
calves
compared
to
non-infected
calves.
From
this
it
can
also
be
concluded
that
the
frequency
of
null
cells
(non-T,
non-B,
non-macrophage
cells)
was
elevated
in
the
infected
calves.
From
the
data
in
Tables
3
and
4
it
can
be
inferred
that
in
the
animals
included
in
this
study,
approximately
20%
of
all
T-cells,
4%
of
the
B
cells,
24%
of
the
monocytes
and
53%
of
the
null
cells
contained
virus
antigen.
Within
the
infected
T
cell
population
BoT8+
cells,
the
suppressor/cytotoxic
cell
phenotype,
accounted
for
approximately
60%
and
the
BoT4+
cells,
the
helper
cell
population,
for
40%
of
the
infected
cells.
Subpopulations
of
PBL
replicating
BVDV
A
variety
of
experiments
were
performed
to
estimate
the
virus
production
by
the
various
subpopulations
of
mononuclear
cells
in
peripheral
blood.
As
infected
cells
occurred
amongst
all
the
major
subgroups
and
each
of
them
contributed
different
numbers
of
positive
cells
and
yields,
it
appeared
exceedingly
difficult
to
obtain
a
clear
picture
of
this
aspect.
However,
as
depicted
in
Table
5,
where
the
different
physical,
chemical
and
immunological
cell
separation
procedures
1976
H.
BIELEFELDT
OHMANN,
L.
RONSHOLT
AND
B.
BLOCH
4
Fig.
1.
Double
immunolabelling
of
PBL
from
a
persistently
viraemic
calf
to
demonstrate
virus
infection
in
phenotypically
defined
cell
subsets.
The
cytospin
preparation
was
stained
for
the
BoT8
and
BVDV
antigens
using
a
combination
of
the
A
PAAP
and
HRP/DAB
systems.
The
BoT8+
cells
and
BVDV
antigen-positive
cells
cannot
be
clearly
distinguished
in
this
black
and
white
photomicrograph.
However,
cells
positive
for
both
antigens
stand
out
distinctly
as
very
dark
stained
cells
(arrows).
Bar
marker
represents
10
1.1m.
significantly
reduced
the
various
target
subgroups
of
mononuclear
cells,
it
appeared
that
monocytes
(expt.
I
no.
K10
and
14,
and
expt.
II
no.
3)
and
T
cells
(expt.
V,
no.
6
and
13
and
expt.
III,
no.
12)
were
able
to
produce
infective
virus.
In
contrast,
B
cells
(expt.
V,
no.
6
and
13)
contributed
very
little
or
not
at
all
to
the
virus
propagation.
The
latter
finding
supported
the
productive
role
of
monocytes
inferred
from
experiments
III
(calf
no.
12)
and
IV
(calves
no.
144
and
6).
BVDV
infection
of
glass-adherent
cells
from
persistently
infected
calves
The
number
of
blood
monocytes
had
significantly
increased
in
persistently
infected
calves
(Table
4)
and
the
cells
appeared
'activated',
i.e.
were
larger,
more
vacuolated
with
more
and
longer
pseudopods
than
in
monocytes
from
non-infected
animals
(not
shown).
The
monocytes
also
constituted
a
large
proportion
of
the
virus-infected
cells,
and
because
of
the
inherent
nature
of
monocytes
to
migrate
to
all
kinds
of
tissues
and
differentiate
into
mature
macrophages,
a
few
studies
were
conducted
to
characterize
this
population
further.
PBL
suspensions
from
infected
and
non-infected
calves
were
seeded
in
a
fivefold
dilution
row
into
duplicate
wells
of
24
well
tissue
culture
plates
with
cleaned
glass
coverslips
and
incubated
for
14
h.
Non-adherent
cells
were
then
rinsed
off
thoroughly
and
the
coverslips
transferred
to
new
trays
or
fixed
for
immunocytochemical
studies.
In
the
former
case,
MDBK
cells
were
seeded
onto
the
macrophage
cultures
and
the
cocultures
incubated
for
4
days
at
which
time
virological
assays
were
conducted.
This
series
of
experiments
revealed
that
whereas
in
non-infected
animals
only
one
cell
out
of
2000
NSE+
cells
in
a
PBL
preparation
adhered
and
developed
in
vitro
into
a
mature
macrophage
(i.e.
0-05%),
one
cell
per
35
to
140
NSE+
cells
would
do
so
in
the
persistently
Table
4.
Frequencies
of
BVDV
in
blood
of
persistently
infected
cattle
1977
mononuclear
cell
subpopulations
in
peripheral
blood
of
non-infected
and
persistently
BVDV-infected
cakes
0
/
0
positive
among
PBL
Calves
persistently
Subpopulation
of
PBL
Non-infected
infected
with
BVDV
B
lymphocytes
19.8
(15.0-30.3)t
210
(14.7-40.0)
(BIg73+/PIg45+)*
(n
=
17)I
(n
=
45)
T
lymphocytes
59.8
(37.5-77.4)
46.8
(31.7-63.7)
(CH128+/CH61a+)
(n
=
14)
(n
=
14)
BoT4
cells
31.3
(29.0-32.3)
31.7
(27.0-39.7)
(IL-Al2)
(n
=
5)
(n
=
6)
BoT8
cells
23.2
(17
,
7-27.5)
19.5
(12.0-29.5)
(IL-A17)
(n
=
9)
(n
=
17)
Monocytes
11.6
(9-0-17.5)
16.5
(8.2-28-5)
(DH59A)
(n
=
6)
(n
-
.=
27)
Monocyte
subpopulation
6.9
(0.0-14.0)
8.5
(3.0-12.5)
(CH137A)
(n
=
5)
(n
=
7)
Monocyte
subpopulation
10.8
(8.5-14.0)
8.8
(1.5-13.5)
(CH16A)
(n
=
5)
(n
=
7)
Monocyte
subpopulation
7.5
(5.0-10.0)
17.5
(10.5-20.5)
(IL-A23)
(n
=
3)
(n
=
6)
Monocytes
(NSE+)
112
(5.0-18.5)
17.8
(11.5-25.1)
(n
=
18)
(n
=
28)
MEIC-H
(DR
equivalent)
32.9
(26-0-41.0)
32.3
(20.9-47.0)
(TH14B)
(n
=
13)
(n
=
33)
MHC-II
(DQ
equivalent)
28.8
(17.0-37.1)
25.7
(16.5-34.0)
(TH81A)
(n
=
13)
(n
=
23)
MHC-II
(DP
equivalent)
31.5
(20.0--40.5)
27.1
(20.2-35.1)
(H42A)
(n
=
11)
(n
=
17)
*
MAbs
used
for
the
detection
of
the
phenotypic
cell
marker
(see
Table
1).
t
Range
of
frequencies.
$
n,
Number
of
separate
determinations
(different
animals
and
different
days).
infected
calves
(i.e.
0.7
to
2.8%).
However,
no
significant
differences
were
found
between
the
adhering
cells
in
the
two
animal
groups
with
respect
to
expression
of
MHC-II
antigens
or
the
macrophage
differentiation
antigens
defined
by
DH59B
and
IL-A23
(data
not
shown).
Another
notable
finding
in
this
series
of
experiments
was
that
whereas
the
proportion
of
virus
antigen-
positive
cells
among
the
adherent
NSE+
population
in
most
cases
corresponded
quite
well
to
the
estimates
previously
made
and
correlated
well
with
the
total
frequency
of
virus
antigen-positive
cells
in
the
PBL-isolates
(Table
6),
only
a
small
proportion
of
the
cells,
i.e.
from
5.0
to
12.5%
of
the
adherent
cells
were
productively
infected
(Table
6)
as
detected
by
cocultivation
with
MDBK
cells.
If
the
monocytes
and
macrophages
were
left
to
mature
in
vitro
for
5
to
6
days
before
cocultivation,
up
to
100%
of
the
cells
could
appear
to
be
productively
infected
(data
not
shown).
Electron
microscopy
Examination
of
ultrathin
sections
of
cell
preparations
by
electron
microscopy,
either
non-
fractionated
PBL
or
enriched/depleted
populations,
including
monocyte
cultures,
revealed
BVDV-like
particles
with
a
size
of
45
to
55
nm.
The
particles
were
found
in
small
cytoplasmic
vesicles
in
both
typical
lymphocytes
and
in
monocytes
(Fig.
2).
In
no
instance
were
virus
particles
found
attached
to
the
cell
surface,
thus
supporting
the
control
experiments
for
non-
specific
virus
'carry-over'
described
above.
By
labelling
subpopulations
of
cells
by
the
immunogold
technique
and
employing
the
MAbs
listed
in
Table
1,
cells
containing
viral
particles
could
be
phenotypically
identified
as
T
and
B
cells
as
well
as
monocytes.
This
corroborated
both
the
results
of
the
immunocytochemical
studies
and
the
findings
presented
in
Table
5.
1978
H.
BIELEFELDT
OHMANN,
L.
RONSHOLT
AND
B.
BLOCH
Table
5.
Virus
antigen
and
infectious
virus
in
enriched
populations
of
mononuclear
cells
from
peripheral
blood*
Expt.
&
animal
number
Treatment
Frequency
(%)
A
%
BVDV-antigen-
positive
cells
TCID
50
/10
6
cells11
T
cellst
B
cellst
I
2
None
66.0
17.3
11.5
0
0
L-LME
72.0
19.5
0.5
0
0
K10
None
55.5
16.0
17.5
15.0
160
L-LME
70.3
10.5
2.5
17.0
5
14
None
55.0
26.3
22.0
25.0
128
L-LME
57.7
24.5
7.5
29.0
20
II
1
None
46.5
27.0
27.0
19.9
1024
Defibrinated
blood
62.5
24.7
<0.7
27.3
NOR
3
None
48-3
324
14.3
27.0
512
Defibrinated
blood
72.7
15.0
2.5
27.5
256
4
None
49.3
27.0
16.8
22.6
256
Defibrinated
blood
69.0
17.7
1.7
25.7
128
III
7
None
45.7
27.0
20.5
30.4
512
Sephadex
NA
71.3
11.7
<0.01
32.9
256
AD
cells
6.7
1.3
80.0
18.1
256
12
None
48.3
22.0
13.3
22.0
128
Sephadex
NA
57.7
16.3
<0.01
30.3
512
AD
cells
7.0
2.3
88.3
24.3
256
14
None
44.7
18.5
26.3
28.8
256
Sephadex
NA
72.4
13.0
1.0
29.8
256
AD
cells
2.7
1.3
94.3
35.0
ND
IV
144
None
31.9
30.5
11.7
24.7
171
C'
control
40.7
29.3
9.3
24.3
160
TH14B+C'
(<1%
TH14+)
60.7
<0.3
<0.3
29.0
40
6
None
43.3
20.3
21.7
15.0
64
C'
control
48.0
22.5
21.3
19.7
64
TH14B+C'
93.0
<0.3
2.3
20.1
32
8
None
56.0
20.7
13.0
0
0
C'
control
58.0
20.7
13.0
0
0
TH14B+C'
83.0
<0.2
<0.7
0 0
V
5
None
52.0
15.1
5.0
0
0
C'
control
60.0
13.5
4.0
0
0
PIg45+C'
86.6
<0.3
4.3
0 0
B26A4+C'
1.3
28.0
ND
0
0
6
None
43.3
28.3
17.3
23.3
128
C'
control
51.7
18.3
14.2
22.3
40
P1g45+C'
67-0
3.0
13.7
19.0
80
B26A4+C'
1.7
38.3
ND
10.0
40
13
None
58.3
26.3
16.3
9.7
128
C'
control
65.5
15.0
12.0
8.3
80
PIg45+C'
73.0
0.3
11.3
11.7
160
B26A4+C'
5.0
34.0
12.0
5.0
20
*
Representative
examples
from
a
series
of
more
than
35
separate
experiments.
Cells
positive
for
the
CH128-defined
antigen.
Cells
positive
for
the
BIg73-defined
antigen.
§
NSE+
cells
(M,
monocytes/macrophages).
II
Infectious
virus
isolated
directly
from
cell
lysates.
ND,
Not
determined.
DISCUSSION
In
this
study
it
was
found
that
in
vivo,
BVDV
infects
and
replicates
in
subpopulations
of
all
the
four
major
cell
groups
in
the
peripheral
blood,
i.e.
T
and
B
lymphocytes,
monocytes
and
null
cells.
Of
these,
the
B
cell
population
seems
to
make
only
a
minor
contribution.
This
finding
is
interesting
in
the
context
of
the
apparent
tolerance
to
the
persisting
virus,
as
defined
by
the
complete
absence
of
neutralizing
antibody
production,
which
characterizes
these
animals.
Several
explanations
for
this
apparent
paradox
might
be
considered.
Thus,
the
virus-specific
x
r
BVDV
in
blood
of
persistently
infected
cattle
1979
}
Fig.
2.
Electron
micrograph
of
BVDV-like
particles
in
a
small
cytoplasmic
vesicle
in
a
lymphocyte
from
the
peripheral
blood
of
a
persistently
infected
calf.
The
cells
were
fixed
in
2.5%
glutaraldehyde
only.
Bar
marker
represents
100
nm.
Table
6.
BVDV
infection
in
glass
-
adherent
cells
from
persistently
infected
calves*
Glass-adherent
cells
BVDV-antigen-
Virus-producing
BVDV*
cells
Calf
as
%
of
NSE÷
cells
positive
cells
(%)t
cellst
(%)
in
PBL
(%)
6
0.7
17.9
62
15.7
K10
1.4
14.4
8.5
16.5
T10
2.8
21.6
62
13.8
13
0.8
8.8
5.0
9.5
14
1.4
24.1
12.5
27.3
*
The
results
are
averages
from
four
or
five
independent
experiments
in
each
calf.
t
The
proportion
of
those
NSE+
cells
which
had
adhered
after
a
14
h
incubation
of
PBL.
lack
of
response
could
be
due
to
an
indirect
effect
on
B
cell
functions
caused
by
a
defect
in
virus
antigen
presentation
by
macrophages
or
in
the
production
of
some
essential
factor(s)
by
macrophages
and/or
T
lymphocytes.
Alternatively,
the
infection
of
T
cells
and
monocytes
could
induce
virus-specific
suppressor
cells
amongst
either
or
both
cell
types.
Indeed,
Larsson
(1986)
did
report
the
presence
of
a
Fc
receptor-bearing
suppressor
cell
in
PBL
from
persistently
infected
calves.
However,
the
possibility
of
a
direct
effect
of
the
BVDV
on
the
B
cells
cannot
be
ruled
out
either.
Thus,
the
very
few
virus-infected
B
cells
encountered
may
represent
vestiges
of
a
much
more
extensive
B
cell
infection,
perhaps
at
the
bone
marrow
level,
causing
clonal
deletion
of
the
virus-specific
(precursor)
cells.
Future
studies
should
be
aimed
at
clarifying
these
aspects.
The
variability
of
the
levels
of
infection
between
individual
calves
was
notable
(Table
2).
The
infection
level
could
not
be
related
to
any
aspect
of
the
health
status
of
the
animals,
as
all
except
two
remained
clinically
normal
throughout
the
investigation
period
stretching
over
more
than
8
months.
In
those
two
calves,
which
succumbed
to
MD,
no
increase
in
infection
level
of
PBL
was
1980
H.
BIELEFELDT
OHMANN,
L.
RONSHOLT
AND
B.
BLOCH
observed
in
or
around
the
time
of
disease
development
and
death
(unpublished
data).
It
is
therefore
by
no
means
obvious
what
kind
of
factor(s)
might
govern
the
persistence
and
replication
of
BVDV
in
the
PBL.
Stimulation
of
the
PBL
with
mitogens
or
interleukin
2
failed
to
enhance
virus
rescue
by
cocultivation
(H.
Bielefeldt
Ohmann,
unpublished
data),
perhaps
suggesting
that
it
is
'fixed'
differentiation
subsets
of
cells
that
replicate
the
virus.
Thus,
cell
division
may
provide
no
or
only
a
minor
contribution
to
virus
persistence,
but
rather
the
infection
is
maintained
by
continuous
infection
of
newly
recruited
cells
within
each
group
of
cell
types.
It
will
therefore
clearly
be
of
interest
to
explore
what
is
happening
in
the
primary
lymphoid
tissues,
i.e.
thymus,
bone
marrow
and
perhaps
the
gut-associated
lymphoid
tissues,
in
the
persistently
infected,
clinically
normal
calves.
Furthermore,
among
the
candidate
factors
with
potential
for
influencing
establishment
and
maintenance
of
virus
persistence
are
the
interferons
(Jacobson
&
McFarland,
1982;
Matthews
&
Vorndam,
1982;
von
Rheinbaben
et
al.,
1985).
Their
role
in
persistent
BVDV
infection
requires
some
attention.
The
demonstrated
activation
of
the
monocyte
population
of
infected
animals,
i.e.
increased
numbers,
enhanced
membrane
ruffling,
vacuolation
and
increased
propensity
to
adherence,
also
among
cells
not
containing
detectable
viral
antigen,
could
potentially
be
due
to
interferon
exposure
(Bielefeldt
Ohmann
et
al.,
1984,
1986;
Bielefeldt
Ohmann
&
Babiuk,
1986a).
However,
the
possibility
of
the
recruitment
of
a
completely
new
subset
of
the
macrophage
lineage
during
the
course
of
infection
should
not
be
ruled
out
at
present
(Narayan
et
al.,
1984;
Gendelman
et
al.,
1985).
Although
based
on
a
relatively
small
number
of
samples
it
was
striking
to
find,
by
electron
microscopy,
virus-like
particles,
similar
to
those
previously
described
in
the
tissues
of
calves
suffering
from
MD
(Bielefeldt
Ohmann
&
Bloch,
1982)
in
a
much
larger
proportion
of
cells
than
that
expected
from
the
direct
isolations
from
the
corresponding
cell
lysates.
This
could
of
course
be
due
to
low
recovery
efficiencies,
either
because
of
suboptimal
sensitivity
of
the
MDBK
cells
used
in
our
isolation
assay
or
due
to
the
presence
of
intracellular
inhibitors
liberated
simultaneously
with
the
intracellular
virus
particles.
Alternatively,
it
might
indicate
that
only
an
exceedingly
small
proportion
of
the
virions
produced
are
actually
infectious,
a
well
known
phenomenon
in
virology
(Richman
et
al.,
1984).
This
could
also
explain
the
low
virus-titres
usually
achieved
with
BVDV
both
in
vivo
and
in
vitro
despite
widespread
virus
infection
(up
to
100%
in
vitro)
as
detected
by
immunocytochemistry
(Horzinek,
1981;
Bielefeldt
Ohmann,
1981).
Finally,
the
infection
of
the
null
cell
population
should
be
briefly
commented
on.
This
cell
type,
which
may
contribute
up
to
20%
of
PBL
in
normal
cattle
(W.
C.
Davis,
personal
communication;
Morrison,
1986;
H.
Bielefeldt
Ohmann,
unpublished
observations
and
the
present
work,
see
Table
4)
appeared
to
contain
up
to
40%
of
all
the
virus
antigen-positive
cells
in
a
persistently
infected
animal,
with
approximately
50%
of
the
null
cell
population
being
infected.
At
least
two,
not
mutually
exclusive,
possibilities
exist.
The
null
cell
population
may
include
precursor
cells
of
the
other
three
major
cell
groups
not
yet
expressing
the
phenotypic
markers
used
for
their
detection.
Alternatively,
the
virus
infection
may
cause
a
suppression
of
normal
cell
markers
(Jennings
et
al.,
1985),
which
then
escape
proper
classification.
Whatever
the
explanations
are,
the
null
cell
population
deserves
more
attention
considering
its
major
contribution
to
the
virus
persistence
in
PBL
of
tolerant
cattle.
In
conclusion,
the
establishment
of
the
identities
of
the
infected
cells
in
persistently
BVDV-
infected
calves
will
now
allow
a
further
elucidation
of
the
mechanisms
governing
establishment
and
maintenance
of
virus
persistence,
including
the
possible
effects
on
the
specialized
functions
of
the
immune
cells,
whether
of
a
direct
nature
due
to
virus
infection
per
se
or
indirectly
by
upsetting
normal
functional
circuits,
as
the
shifts
in
relative
frequencies
(Table
4)
may
indicate
(Woodruff
&
Woodruff,
1975).
The
authors
thank
Dr
W.
C.
Davis
(Washington
State
University,
Pullman,
Wash.,
U.S.A.)
and
Dr
A.
J.
Teak
(ILRAD,
Nairobi,
Kenya)
for
the
generous
gift
of
MAbs.
The
provision
of
BVDV-free
MDBK
cells
and
the
access
to
animals
made
possible
by
Dr
A.
Meyling
(The
National
Veterinary
Laboratory,
Copenhagen,
Denmark)
are
greatly
appreciated.
Thanks
are
also
due
to
Helle
Kurstein
and
Irene
Kosokowsky
for
typing
the
manuscript.
Financial
support
was
received
by
H.
Bielefeldt
Ohmann
from
the
Danish
Agricultural
and
Veterinary
Research
Council
(grant
no.
SJVF
13-3681).
BVDV
in
blood
of
persistently
infected
cattle
1981
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