1 - Blood Journal

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Feline
Parvovirus
Propagates
in Cat
Hematopoietic
By
Feline
Gary
J.
parvovirus
infected
cats.
(FPV)
We
poietic
depression
cultured
in vitro.
FPV
and
Viral
hybridization
in
Granulocytes
and
infected
cultures
viral
capsid
of
were
six days.
assays.
IRUSES
MAY
aplastic
anemia
at
the
Aplastic
inactivation
neutralizing
‘5
was
depressed
also
the
factors
the
etiology
evidence
of
for viral
rus,
and
sion
in their
is clinically
FPV
in the
by FPV.
& Stratton.
anemia
in persistently
infected
immunosuppressed
persons.5
Although
anemia
and reticulocytopenia
are the
most prominent
hematobogic
features,
B 19 parvovirus
also
to substantial
rus
has
specific
been
and
toxic
Parvoviruses
Parvoviruses,
extremely
reduction
in beukocytes
and experimental8
shown
in cell
erythroid
genome,
platelets
progenitor
are common
agents
small DNA-containing
limited
and
in
and
that
can
cells.9”#{176}
parvovirus
replica-
type.’3”4
The bone
with a wide variety
differentiation,
parvovirus
provides
replication.
Panleukopenia
most
present
study,
we
feline bone marrow
investigated
cells. The
of virus-associated
marrow
hematopoietic
were
shown
to feline
to be infectious,’7
period
followed
emesis,
anorexia,
rally
infected
profound
parvovirus
diseases
and Enders
as well
failure
was a prominent
FPV infection
is clinically
rhea,
the
organ
stages
of
animals
for
is one of the
in Europe
and
the
and
over
animals,
50 years
death
may
manifested
tenderness.’5
result
from
In experimentally
with
typical
DNA
disease.
sequence23
Blood,
Vol 74. No 1 (July),
ago
Ham-
Celilines
and
feline
kidney
were
maintained
cells
(from
in
com-
marrow
depres-
the interaction
of
feline model is useful
failure:
and
are
feline
hema-
the animals
Our
studies
direct
targets
can be
indicate
of infection
were
in
Biofluids)
Infectious
of
NLFK
Virus
plaque
assay.
cells
added
incubator
(5%
CO2,
in prewarmed
virus
served
which
and
were
Cells
the
Cancer
serum
(FEF,
Center,
modified
Eagle’s
and incubated
two
FPV
medium
to three
days
(strain
as
after
CU-4,
in supernatants
tissue
and
of
first
bow
and
culture
dishes
(Becton-
37#{176}C).Medium
(DMEM
of successive
to each
virus
medium
incubated
was
in the
with
‘/,o dilutions
plate;
were
inoculum
a
4 to 8 x l0
overnight
3 mL
NLFK
procedure,
to attach
added
virus
the
trypsinized
and allowed
aspirated
as control.
after
Kettering
In
was
humidity,
media
fibroblasts
10% FCS
methods.
plastic
NJ)
95%
was then
virus
hours
Park,
medium
calf
embryo
virulent
cells
100 mm
x
Lincoln
Eagle’s
5% fetal
obtained
of FPV
two
of FEF
to 20
Dickinson,
University)
Dr Carmichael.
Titers
by
monolayer
with
from
determined
confluent
with
were
cells
obtained
were
Dulbecco’s
supernatants
strain)
cells
Sloan
supplemented
passage
of
containing
37#{176}C.
Feline
of Crandell
Cornell
modification
MD)
Memorial
maintained
(DMEM;
above.
Hardy,
derivative
Carmichael,
Iscove’s
in 5% CO2.
Dr William
NY)
cells, a cloned
Dr Leland
Rockville,
Biofluids)
from
NLFK
virus.
cells
Biofluids,
From
as others
showed
that bone marrow
feature
of the disease.’8’2#{176}Natural
characterized
by a brief incuba-
by gastroenteritis,
and abdominal
neutropenia.
parvovirus
(FPV)
of cats
animals,
FPV does not result
in clinical
nia is a consistent
finding,2’
suggesting
of
in adult
FPV
of
without
for six to I 2
removed
by
careful
This clinical
syndrome
was one of the first
attributed
to a virus after cell free extracts
mond
tion
proliferating
lineages
and
target
and
METHODS
10% FCS)
infectious
States.’5”6
diseases
a continually
of various
a good
due
important
United
animal
marrow,
of cells
col-
supported
formation.
produce
species.24’25
inoculation
tion is dependent
on actively
proliferating
host cells.’2 These
viruses
have a propensity
to infect
fetal tissue,
leading
to
premature
abortion
or malformation.”
Parvoviruses
also
may show
specificity
for a stage
of differentiation
or cell
also
host
feline
(FCS,
of animal
disease.”
animal
viruses,
have an
therefore
G-CSF
colony
can be analyzed
in vitro
experimentally
manipulated.
(IMEM;
infection.
The Bl9 parvovicultures
studies
to be highly
for human
with
human
Inc.
parvoviruses
of chronic
both natural6’7
by
assays.
dependent
appropriate
study
termed
B19
sient aplastic
identified
as the cause
of tranwith underlying
hemolysis4
and
these
the
with
In
episode
of infecof the immunologic
topoiesis
readily
leads
of
be abrogated
preincubation
and
progenitor
factor
by Grune
or
and
presence
Recombinant
GM-CSF
in
inhibited
the
could
virus
cells
(BFU-E
in
formation
of
progenitor
formation
antibodies.
myelopoiesis
1989
by myeloid
colony
stimulating
pletely
formation
erythroid
of colony
specific
abnormalities
seen in patients
with aplastic
anemia
are also
observed
in individuals
with chronic
retrovirus
or herpes
virus infections.3
Recently,
one of the family of Parvoviridae,
parvovirus,
was
crisis in persons
colony
but
physical
feline
absent
showed
and Neal S. Young
Frickhofen,
Inhibition
ony
Inhibits
inhibited
anemia
cases with a preceding
or hepatitis.”2
Many
3.
by immunofluo-
to
but
day
cells
efficiently
CONTRIBUTE
is circumstantial.
associated
in some
tious mononucleosis
most
in humans,
by in situ
virtually
Infected
in nuclei
FPV
virus.
molecular
cells
Norbert
CFU-E-derived)
demonstrated
DNA
Lustig,
and
In Vitro
(CFU-GM).
marrow
observed
marrow
predominantly
In clonal
we
were
precursors
after
bone
Cultures
hematopoietic
naturally
of hemato-
feline
using
DNA
1 0%
their
protein
in
cultures
and
about
from
causation
virus
Marrow
Formation
Lawrence
in
mechanism
replication
RNA
Platanias,
leukopenia
the
In suspension
propagation
V
causes
this
Colony
Leonidas
investigated
by
hybridization.
rescence.
Kurtzman,
Bone
other
sepsis
infected
to
germ-free
illness,
but neutropea possible
interaction
microorganisms
pp 71-81
due
the Cell
Heart,
Submitted
as diarIn natu-
in producing
FPV is closely
related
antigenicably22
to mink enteritis
virus and canine
1989:
National
and by
parvovi-
Biology
Lung,
Section.
and
September
Blood
29.
Clinical
Institute,
1988;
accepted
An abstract
ofpart
ofihis
work
1985
and Blood 72:504a, 1988.
Address
reprint
Hematology.
requests
Stanford
Stanford,
CA 94305.
The publication
costs ofthis
charge payment.
This article
article
must
“advertisement”
with
indicate
© I 989
in accordance
24.
1989.
in Blood 66:134a,
J. Kurtzman,
School
Branch.
MD.
February
was published
to Gary
University
Hematology
Bethesda.
MD.
ofMedicine.
Division
Room
of
5161,
were defrayed
in part by page
therefore
be hereby
marked
18 U.S.C.
section
1 734 solely
to
this fact.
by Grune
& Siration.
Inc.
0006-4971/89/7401-0024$3.00/0
71
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72
KURTZMAN
washing
with
fluids).
The
0.5%
prewarmed
methylcellubose
hours.
the
After
were
for
carefully
St
floated
NaCl
for four
to remove
mol/L
minutes
sodium
five
and
was performed
below.
using
Viral
“plaques”
mob/L
was
Screen
filters.
Individual
for four
minutes,
Tris-HCI,
pH
pH
Organon
7.5,
(Hyclone,
PBS,
desired
amount
(New
volume
of uninfected
tration
was brought
and virus
four
were
1.5 mob/L
incubated
for
wiping
of the filters
with
were
then
washed
in 2 x SSC
(0.3
same
cell
sodium
citrate)
for at least
two
parvovirus
(CPV)
autoradiography
by
(Fig
remained
with
hours.
probe
technique,
for
more
poietic
of
the titer
than
6 months
room
and
temperature
with
incubated
parvovirus
capsid;
after
capsid
England
of dependence
reagent
of bone
iones,
mixture
Laboratories,
marrow.
(ketamine)
aspiration
0.05
mL
and
20
syringes
medium
Feldman,
into
U/mL.
of sodium
of
MD)
(immune
(IMDM;
St Louis).
syringes
diatrizoate-Ficoll
in-2
but
and
lack
specific
tibia,
1 mL
GIBCO,
heparin
(I .077
were
under
of Iscove’s
Grand
heparin
cells
and
chloroform
general
modifica-
Island,
U/mL;
NY)
concen-
separated
to 1.080
with
g/mL;
LSM,
incubating
in 10 mmol/L
at 4#{176}C
until
conditions
a
and
analysis,
to 150
.tl in TE
NaOH
and
neutralized
samples
and
dot
briefly
washed
at
0.5%
SDS
with
shaking
blot
for
NaCI,
0.5%
were
was
Tris,
200
SDS,
India-
by phenol
Cells
1 mmol/L
and
were
EDTA
extracted
of tRNA
or supernatant
addition
resus-
(TE)
and
under
the
but
30 minutes
and
were
Schuell,
Keene,
baked
then
prehyridized
were
then
solution)
incubated
ammonium
and
and then
5 x Denhardt’s
diluted
not
for
NH)
were
then
acetate
and
Filters
hours
in
buffer
to four
in
x SSC)
on a 96 well
for two
overnight
of 3 N
(10
Schuell).
two
to 100
volume
to preequilibrated
(Schleicher
(6 x SSC)
1/10
at 65#{176}C.
Samples
of 2 mol/L
applied
were
of
were
in a vacuum
(6 x SSC,
hours
buffer
at 65#{176}C
with
100
io-
Fig
Three
dilutions
in-5
virus
Biochemicals,
the addition
volume
manifold
and
were
20#{176}C.
-
in 10 mmol/L
precipitation.
by the
dilutions
80#{176}C.Filters
cells
at
Cell-associated
pH 7.5,
A
Sweden).
and
pellets
pellets
mmol/L
pellets
(Schleicher
DNA
oven
cell
an equal
protein
analysis.
followed
incubation
with
and
times
as cell
Supernatants
before
peripheral
(Advanced
at 37#{176}C
followed
or without
hemato-
Uppsala,
Mannheim
Tris-HCI,
with
FCS
desired
cell
ethanol
assay.
precipitated
DNA
100
media
cat
RPMI
in
virus.
overnight
extraction
nitrocellubose
from
EDTA,
stored
then
O’Neil,
was collected
at a final
by
of
at the
stimulation,
and stored
K (Boehringer
pended
For
as
the
Cells
10% human
stimulate
10%
and
humidity.
resuspended
Pharmacia,
the washed
1 mmol/L
that
with
at
identification
proteinase
same
pathoserved
or humerus
(1,000
blood
Ni)
and
7.5,
ethanol
Corners,
femur,
containing
results,
serum).
Peripheral
Mononuclear
A
harvested
in medium
by resuspending
napolis),
FPV
determined
simplicity
was obtained
the
containing
free
was
to I -year-old
marrow
the
were
times
Cells
FCS
with
resuspended
50 jzg/mL;
pH
95%
and
lymphocyte
aureus,
g/mL
1251 protein
identical
of its relative
20#{176}C),
-
were
FCS,
hours.
conditioned
factors
Columbia,
Isolation
at
anti-canine
with
with
plaques
Liberty
either
of preservative
vein
of
Bone
from
into
of Dulbecco’s
the jugular
tration
(Liberty
to
of
reacts
yielded
Six-month
cultures.
a source
with
number
on a limited
cats
tion
cross
incubation
procedures
I 5 minutes
dilution
After
the
for
(precooled
1 : 1,000
(which
because
Suspension
direct
a
Carmichael).
Both
incubated
acetone-PBS
with
preferable
gen-free
anesthesia
60%
Nuclear),
was
washed,
antiserum
Dr
autoradiography.
the former
then
overnight
from
(New
were
For
cells
four
supplemented
(Staphylococcus
isolated
monolayers
below).
the cell concen-
in IMDM-bO%
stimulated-lymphocyte
(see
three
cells,
in IMDM-b0%
in medium
contains
the
or an equivalent
at 37#{176}C,
5% CO2.
times
FCS
cultures,
at 4#{176}C
for
cells/mL
Biotechnology,
washed
with
manufac-
IMDM-2%
supernatant
in medium
mononuclear
days.
The
three
which
Supernatants
marrow
to cells
1.5 to 2 x106
washed
at 4#{176}C.The second assay, similiar
to a described
technique,26
involved
incubation
of NLFK
cells
with
infectious
supernatant
followed by incubation
ofcells
in media plus methylceblubose
for four
For
UT).
to attach
concentration
cells
blood
in
was mixed
16 to 24 hours
(PHA-LCM),
number
medium
to the
washes
to 2.5 to 5 x iO cells/mL
phytohemagglutinin
as described
the
1 ). By this
SDS
Hybridization
counting
constant
0.1%
to
according
three
as infectious
was allowed
diluted
NC)
by
Logan,
of virus
filters
for
Durham,
followed
Laboratories,
by careful
then
Corp.
directions,
to 96
air dried
7.5,
Teknika
turer’s
were
determined
in supernatants
72
prewarmed
Tris-HCI,
mol/L
a canine
titer
after
of virus
in 0.5
Bio-
containing
incubated
Colony/Plaque
1 mol/L
dried
(PBS;
media
with
nylon
onto
0.03
air
and
0.5 N NaOH
Filters
chloride,
minutes
using
followed
debris.
saline
with
Louis)
82 mm
onto
and immersed
buffered
overlayed
and washing
removed
floated
tissue
for
(Sigma,
Boston)
minutes,
minutes,
then
of media
were
Nuclear,
five
were
aspiration
monolayers
England
phosphate
monolayers
ET AL
1
10
-
1.
FPV
milliliters
(upper
plaque
assay.
of succesive
right
of each
panel) of infected
supernatants
were
incubated
with
permissive feline
embryo
fibroblasts
(FEF). After 4 days. the monolayers were transferred
to nyIon
cellulose
membranes.
lysed, and hybridized
with Plabeled
pRCV.
which
detects
the FPV genome.
The amount
of virus was determined
to be 1
to 2 x 10 plaque forming
units
(pfu)ImL.
From www.bloodjournal.org by guest on February 3, 2015. For personal use only.
FPV
INFECTION
OF CAT
BONE
MARROW
ig/mL
denatured
salmon
sperm DNA
a 32P probe,
labeled
by nick translation
able
kit
Unless
(Bethesda
otherwise
2.3 kb XbaI
Peter
from
Tattersall,
map
units
with
the
same
high
stringency
Virus
known
amounts
FPV
were
performed
New
CPV
overnight
SDS)
at
autoradio-
by comparison
DNA
transferred
Nuclear).
hybridization.
was performed
with
was
to a nylon
The
FPV
EcoRl
specific
stringency
electro-
with
wash
than
Carlson
mately
80%
of
the
ethanol
rehydrated
were
with
100 zg/mL
slides
were
0.1 mol/L
and
then
8.0,
0.25%
ide,
the
and
then
l0
mol/L
performed
as
I mg/mL
salmon
using
with
the addition
l0
cpm
10 mmol/L
DNA.
sealed
overnight
SSC,
50 formamide
Eastman
was
Parafilm
were
in
with
temperature,
stopped
five minutes
in Rapid
bone
marrow
as well
(before
the
cells
of
37#{176}C,
95%
were
incubated
exposed
for
for
three
for
30
Five
in
progenitor
cells
The
included
CT)
and
incu-
(G-CSF,
at 42#{176}C
in 2 x
ethanol
24
washes.
in water;
hours
minutes
acid,
at
and
room
fixed
factor
for
to detect
out
similarly
FPV
in
from
as for the B I 9
human
CHO
obtained
as a pathogen31)
preabsorbed
fixed
blocked
were
against
cat
in 1968
before
the
Dr
red
and
with a
sera for 30 minutes
at
with 0.3% BSA, slides
37#{176}Cwith
a ‘/ai
rabbit
specific
dilution
fold
for
for
1 x
promoting
activity33
l0
from
Genetics
interleukin-3
Institute),
factor
‘/soo,ooo [as
medium
assayed
from
in the
feline
Abkowitz,
and
(M-CSF
fibroblasts
Dr ianis
CA),
stimulating
Genetics
embryo
factor
Oaks,
U/mg;
stimulating
activity
to culture
experiments
stimulating
human
U/mg;
feline
[see below]).
Thousand
Conditioned
burst
was also used
This
enrichment
colony
colony
maximal
Institute).
experiments,
culture.
these
colony
Amgen,
106
the
the
leuke-
(a source
University
at a final
concentration
of 7%.
human
erythropoietin
(epo;
recombinant
at a concentration
of 1 U/mL.
RESULTS
for 30 seconds
at - 20#{176}C
for two hours in PBS with
(FITC)-labeled
Liale, IL) to detect
coli 2 x
and
at 4#{176}C.
The
before
used
derived,
15
for one
to I x 106 cebls/mL,
recombinant
infected
Amgen)
factors
and
for
g/mL),
overnight
washed
granulocyte
cell
was added
1.080
of 30% FCS
immunofluores-
washed in PBS, and incubated
at
from
to
in the presence
(FeLV-A)
some
and
MA),
half
cells
FCS
ratio
two
virus-A
ofcat
incubation
macrophage
cells,
Genetics
For
dog serum
CHO
at 37#{176}C,
resuspended
(1.077
granulocyte-macrophage
Escherichia
from
recombinant
gently
resuspended
protein;
Cambridge,
were
hours
adherence-depleted
with
growth
U/mg
(GM-CSF,
(IL-3,
mia
l0
for two
approximately
human
human
Institute,
4#{176}C,
at
2 x
recombinant
an
to 2 x
monocytes
in IMDM-lO%
and
separated
human
recombinant
then
on LSM
(as assayed
recombinant
then
normal
were
minutes
resulted
at 400
resuspended
the
to plastic
centrifugation
Sweden)
at a 1:1 (vol:vol)
were
washed
procedure
were
and
bone
of a previ-
at 60 g, and then incubated
was repeated
were
mouse];
or control
nonimmune
After
washing
in PBS
fluorescein
isothiocyanate
(ICN
Immunobiologicals,
mixture.
dilution
in 1% acetic
tech-
were
T cells,
DNA-
or eliminate
by
Upsaba,
FCS,
cat).
(Zeiss).
recombinant
separated
erythrocytes
separated
cells
slides
were
(1:1
nonrosetting
Laboratories)
in graded
NTB-3
pathogen-free
5% BSA at room temperature,
fraction
of Washington)
preparations,
frozen,
were
of immune
humidity.
primer
10
To remove
cells
cells
serum, which reacts with the capsid
CPV
Sera
a specific
cence. Cytocentrifuge
in acetone
and stored
‘/,o dilution
Washes
was carried
immune
appearance
from
Greenwich,
Ci/mmob;
(Kodak).
as nonimmune
Carmichael.
cells
the
Immunofluorescence
parvovirus.#{176}Anti-CPV
of FPV,
random
Research
(Kodak)
Fixer
Immunofluorescence.
infected
the
(3,000
Can,
for 30 seconds
4#{176}C.The
mononuclear
and
NY),
developer
at
nonrosetting
in the
on day 8, and
in the marrow,
cells
of adherence
108 rat
in preliminary
a modification
were
washing,
at 37#{176}C,
centrifuged
and the rosetting
by drying
Rochester,
D-19
hour
populations
MidWillow-
activity
to minimize
to I to 2 x 106 cells/mL
with
specific
to the incubation
emulsion
minutes
cell
with
5% CO2.
and 0.5
humidity.
followed
coated
resuspended
(DTT),
labeled
per slide,
(American
humidity,
were
to high
by
Dli’
95%
washes
dextran
IMDM
1% BSA,
microscope
Pharmacia,
After
of virus
Connaught,
of specific
with
and
a final
(Dow,
(shown
and
Cells
g/mL;
by two cycles
incubated
formam-
10%
in
III,
an inverted
purified
cats
at
Laboratoaries),
methylcellulose
factors
of accessory
g, 20#{176}C
for 20 minutes.
from
Cells
on day 5, CFU-GM
using
were
human
in dilutions
promoting
the effects
(1.070
burst
for four hours at 4#{176}C
PHA-LCM
growth
effects
was
(Bethesda
added
at 37#{176}C,
95%
Kodak,
developed
kit
in 50%
(BSA),
10 to
Percoll
(Fluka,
ethanol
a35S-dCTP
IL)
of 20 mmol/L
bated
Slides
Heights,
a commerical
with
graded
through
incubated
burst
were determined
(gran-
aspirated
(step
human
assays
erythroid
to grow
above.
(Hyclone
adequate
lamp
CFU-E)
used
supernatant
0.8%
marrow cells were further
ously described
method.32
dithiothreitol
Probe
with
pH
10%
hematopoietic
removed
temperature.
FCS
to determine
106 cells/mL
for 30 minutes,
formamide
albumin
tRNA,
pH
triethylamine,
was performed
serum
all
by treatment
at room
in
treated
then
was
were
xenon
cell,
to those
erythropoietin
and
the possible
Tris-HC1,
glycine
Hybridization
sperm
of probe
were
was followed
dried
derived
in 30%
CFU-FM;
forming
marrow
or control
to have
In order
at 4#{176}C
in 70%
and
mol/L
Jona-
Cytocentrifuge
of the slides
minutes
ONT),
experiments
approxi-
15 minutes,
bovine
Arlington
nique
Leland
were
(4 to 5 x l0 cpm/ig)
Amersham,
white
ten
dale,
an XBO
of fluorescein.
as described
106/mL
1 U/mL
on days
Dr
contains
in 2 x SSC/50%
slides
1%
mg/mL
x
incubation
NY),
SSC,
This
MI),
BFU-E
from
Bone
gallate
photomicroscope
Progenitor
colony
2-mercaptoethanol,
land,
used
cell,
identical
as a control.
5% n-propyl
III
cultures.
prepared
of 2 x
plated
which
forming
conditions
supernatant
1%
a Universal
erythroid
cells
containing
with
IgG
with
for epifluorescence
colonies.’0
was the ca. 4 kb
K in 10 mmol/L
of 0.1
for
marrow
numbers
genome.
7.0, 0.1 mob/L
anhydride
and
sulfate,
activity
proteinase
in a mixture
acetic
x
for
pH
under
mounted
colony
BFU-E;
CFU-E
and stored
for 15 minutes.
hybridization.
2
FPV
Some
with
acetylation
Ronkonkoma,
the
A (Sigma)
Tris-HC1,
washing
of
cell,
anti-mouse
dry,
FRG)
filters
colony
techniques.27
a gift
which
in 2 x SSC.
RNAse
CaCI2
with
experiments
pEH2O,U
University,
side
forming
32P
described
in 4% paraformaldehyde
incubated
7.5, 2 mmol/L
for these
plasmid
State
left
fixed
before
the
of Colorado
preparations
After
used
from
Zeiss
concentration
of cat bone marrow
of previously
examined
hematopoietic
sulfate,
10% dextran
In situ hybridization
probe
fragment
OberKockem,
bone
membrane
Hybridization
NaCI,
high
by modification
(Carl
performed
above.
In situ
and
Zeiss,
rabbit
blotted
in glycerol,
mononuclear
in 1 mol/L
at 65#{176}C,and
FITC-labeled
washed,
ulocyte-macrophage
washed
before
as described.5
gel and
was
0.1%
were
Clonal
homologous
were
or with
Slides
and standard
DNA.
England
probe
Filters
(from
escence
sequences
(>95%
by densitometry
viral
blots
genome).
was the
pRCV-V
includes
genome
x SSC,
of cloned
on a I % agarose
(GeneScreen;
SDS
of the
which
CPV
MD).
for hybridization
University),
was quantitated
Southern
labeled
used
of the plasmid
(65#{176}Cin 0.1
graphy.
phoresed
the probe
Yale
region
Gaithersburg,
fragment
20 to 65 of the
73
and 2 to 4 x 106 cpm/mL
of
with a commerically
avail-
Laboratories,
indicated,
approximately
Dr
Research
IN VITRO
of
anti-dog
IgG
immunofluor-
FPV
tures.
cells
propagation
Suspension
obtained
from
in
of bone
specific
pathogen-free
bated
with
FPV,
and
inocubum
was removed
by dot blot
time
course
suspension
cultures
bone
marrow
marrow
cats
cul-
mononuclear
were
innocu-
after
an overnight
incubation,
the
by washing.
FPV DNA
was detected
analysis
with a CPV-labeled
experiment
showing
the
probel.
appearance
A typical
of viral
From www.bloodjournal.org by guest on February 3, 2015. For personal use only.
74
KURTZMAN
DNA
in cells and supernatant
in Fig 2. In general,
blood
of a cat bone marrow
culture
cell associated
FPV
DNA
is shown
appeared
within
24 hours of inoculation,
and virus in supernatant
was detectable
with a variable
delay (24 to 48 hours),
depending
on
associated
with
cultures
was
3).
results
The
the
donor
cells
linearly
cells.
and
rebated
of virus,
not
reached,
the
size
target
a plateau
of
cells
the
virus
was
limiting.
under
inoculum
rather
quantity
subsequent
virus.
of virus present
at later
cycles
of reinfection
in animals
A
than
supernatant
of target
was
number
3 after
virus
rather
effects
than
of two
propagation.
(we
in
the
not
possibility
in T cells
hematopoietic
within
cells,
of cell
A is a mitogen
confirmed
were
examine
occurring
stimulators
Protein
lymphocytes35
lectin
by the
we
proliferation
for circulating
that
concanavalin
A and
efficient
stimulators
of lym-
shown),
the
of
of
FPV
the
the
cultures
in the
replication
increased
in
of
was
Addition
which
and,
of
stim-
under
marrow
erythropoietin,
hormone
(never
more than
on viral replication
4).
medium
propagation
on viral
medium
experiments,
modestly
no effect
A (Fig
of FPV
to enhance
addition
conditioned
in
in marrow
propagation
failed
effect
of protein
leukocyte-conditioned
hematopoiesis
conditions,
some
presence
stimulated
ulates
of
infection,
numbers
the
inhibited
in culture
indicated
newly
synthesized
lymphocyte
marrow
was
phocyte
proliferation;
data not shown).
FPV propagation
cat peripheral
blood cultures
was increased
by the addition
protein
A, but FPV propagation
in cat bone marrow
FPV DNA
was
cells.
The
larger
times
with
reduces
of
conditions
the
at day
bone
in vitro
phytohemagglutinin
(Fig
DNA
the
cat
of target
maximal
culture
nodes.’5”6”82#{176} To
propagation
on
of
24 hours
FPV
these
In contrast,
of cell-associated
and
suggesting
depletion
infection
after
in cell-associated
that
inoculum
lymph
FPV
compared
DNA
supernatant
cycle of infection
with
addition
of
a plateau
reached,
FPV
of viral
the
performed
a single
Even
suggesting
amount
in
to the size of the
of experiments
culture
likely
represent
cells
by input
virus.34
amounts
The
present
and
that
ET AL
certain
(data
not
in the blood.
The
with
was
or
without
inconsistent:
virus
in
production
two-fold),
but in others
(data
not shown).
there
was
w
4
/
.
.,..
,
V
I
U)Q)
-.--
E
o.
C
1O
C)
C
.
>1
U)
lc
Cc’,
I
j5Q)
I
108
01
I
day
0
1
B
I
I
I
I
2
3
4
5
pRCV
C
2x105
2x104-
:
8,
C.)
2x103-
-
#{149}:41,
S
1
10
._1
Fig 2.
C’,
50-
I
=
0.51
e
I
!
course
of FPV
infection
in suspen-
sion cultures
of cat bone marrow.
Bone marrow
mononuclear
cells were infected
at a virus to cell
ratio of 1 :20 and the amount
of DNA at each time
determined
by DNA dot blotting
of cells and supernant (B). Uninfected
cells (c) and supernatant
after
washing
out the viral innoculum
served as negative
controls.
Positive
controls
included
cloned
viral
DNA (pRCV)
and infectious
viral supernatant
(vi-
Al
rus). The number
of single stranded
were
determined
by densitometry
A. “w” denotes
the time at which
washed
free of the viral inoculu.
virus
C’,
Time
ng
viral genomes
and plotted
in
the cells were
From www.bloodjournal.org by guest on February 3, 2015. For personal use only.
FPV
INFECTION
OF CAT
BONE
MARROW
IN VITRO
75
Replicative
forms
inoculation,
peaked
were
apparent
as early
at around
as 24 hours
48 hours,
and
after
disappeared
by
five days.
FPVin
tion
cence
for
total
ment
Cl)-
cells.
for virus
FPV
DNA
capsid
was also
and
RNA
proteins
detected
(Fig
(Fig
by in situ
6) and
7).
hybridiza-
by immunofluores-
By the
in situ
technique,
FPV nucleic
acids were maximal
at 72 hours.
Pretreatof the slides with RNase
A showed
that the peak of
013
RNA
not
quantity
preceded
shown).
marrow
At
cells
that
peak
showed
were
in infection
infection,
while
the
grains
late
plasmic.
Similarly,
points,
cells
of
20%
(data
of total
deposition
found over the nuclei
of cells,
deposition
was primarily
cyto-
capsid
was
were
one day
about
dense
was
fluorescence
20%
by about
infection,
mainly
grain
viral
immunofluorescence,
of DNA
of
protein,
maximal
mainly
infected
of grains.
in
as determined
at 72 hours.
nuclear.
as
Early
Between
judged
by
by
At all time
10%
the
and
fluorescent
signal.
e)
0
Specificity
Cl)
108
cells
cells
supernant
day 1
day 3
. day 3
-
s--s
#{149}---
ofFPVfor
marrow
suspension
number
of cells
present
of myeboid
lineage
were visible
on
and cultures
contained
predominantly
of
10-5
10-4
10-3
10-2
10-1
Fig 3.
Virus output
in cat bone marrow
suspension
culture
as
a function
of the viral
inoculum.
Varying
amounts
of virus
expressed
as the amount
of virus (pfu) per bone marrow
mononuclear cell were incubated
with cells. After 24 hours. the cells were
washed
free of virus. The number
of cell-associated
viral genomes
was determined
at days 1 and 3 after infection
and the titers in the
supernatant
at day 3 by DNA dot blotting
followed
by densitometry.
replication.
gel
Southern
analysis
DNA
single-stranded
stranded
bar weight
Virus
intermediate
species
replication
(Fig
5A).
viruses,
forms
on agarose
that
gel
was
demonstrated
Parvoviruses,
by
through
migrate
as higher
electrophoresis
virus
was
to demonstrate
pronase
or boiling,
with ribonuclease
a significantly
5B).
of the
inoculum
c?’
by
phase
serum
(Fig
FPV (L. Carmichael,
but not to heating
or deoxyribonuclease
to 56#{176}C
or to treatment
(data
not shown).
mation
comparison
and myeboid
of the dose
(CFU-GM)
greater
compared
response
colony
susceptibility
with
erythroid
of early
formation
of myeloid
to inhibition
erythroid
showed
colony
by FPV
protein
A
control
(10%
peripheral
-
FCS)J
protein
A
control
(1O%FCS)
xlO5cells
for(Table
1
hPHA-LCM
c
of
by
1984).
Colony
inhibiting
of the viral inoculum
with
hPHA-LCM
Fig 4.
Comparison
of FPV
propagation
in blood and bone
marrow
in vitro. In this experiment,
blood or bone marrow
mononuclear
cells
were
infected
with virus at a virus to
cell ratio of 1 :100 in the presence or absence
of protein
A (a
lymphocyte
stimulator)
or
hPHA-LCM
(a source
of hematopoietic
growth
factors).
FCS
alone served as a control.
Cells
were
harvested
at day 3 and
the amount
of virus determined
by DNA dot blot.
on
In the presence
of large quantities
of virus,
essentially
all
myeloid
and erythroid
colony
formation
was suppressed.
molecu-
are
for
effect
was a potent
inhibitor
(Fig 9). Inhibition
convalescent
neutralizes
communication,
May
was sensitive
to treatment
8).
medium
a virus
by the neutralization
with canine
cross reacts
and
10), which
personal
activity
total
Wright’s-Giemsa
macrophages,
erythroblasts
(Fig
assays
in semisolid
in order
abrogated
preincubation
However,
(BFU-E)
(Fig
occasional
colony
marrow
double-
which
replicate
control
hematopoietic
progenitor
cells. FPV
feline
hematopoietic
colony
formation
virus/cell
FPV
bone
of
the
after
1 week of culture
to between
cultures.
By days 6 to 7, very few
cells
stain,
20%
inoculation
reduced
and
feline
10-6
FPV
cells.
consistently
10%
lymphocytes,
and
We established
107
myeloid
cultures
-
bone
marrow
blood
From www.bloodjournal.org by guest on February 3, 2015. For personal use only.
76
KURTZMAN
0
B
a)
A
ET AL
C.)
4
C
9-.
.
ICLO-1r;:1
>‘>>‘
Co
3’
Cl)
CO
2
>‘
CO
CD
DDD
E
5
D
F
-23.1
dimer
RF
3’
5’
9.4
-
-6.6
ss
viral
DNA
single-stranded
viral DNA
CT
‘
monomer
RF
5’
c__
3’
-
4.4
-
2.3
-
2.0
-
1.4
-
1.1
monomer
RF
ci
“2
3’
5’
+
3’
+
dimer
RF
__.)
-
0.87
-
5’
-
+
Fig 5.
Southern
blot of cat bone marrow
cells infected
with FPV in suspension
culture.
Cat bone marrow
mononuclear
cells were
infected
at a virus to cell ratio of 1 :20 and cells were harvested
at varying
times for DNA. The DNA (equivalent
to 2 x 1 0 cells) was then
subjected
to Southern
blot analysis
in order to determine
the presence
or absence
of replicative
forms (A). DNA extracted
from infectious
supernatant
(virus) served as a positive
control.
(B) Replication
scheme
of FPV. FPV. which primarily
packages
the negative
DNA strand in
virions.
replicates
by self-priming
from
the 3’ terminal
hairpin.
Double
stranded
monomer
and dimer
replicative
forms
(RF) are
subsequently
cleaved
to form new single stranded
(as) viral DNA.
1). Late
erythroid
colonies
derived
from
equally
susceptible
to inhibition
as were
The effect
of FPV on cells dependent
factors
was
minimize
cells, cat
and
also
used
or eliminate
bone marrow
rosetting
with
tion
of
exogenous
the
use
of low
as a measure
undefined
percentages
there
was little
of exogenous
growth
medium
and
were
about
colonies.
growth
specificity.
growth
the
factors
calf
colony
factors,
recombinant
was
Under
formation
including
GM-CSF
by
these
without
the
conditioned
and
G-CSF,
suggested
Our
marrow
also
supported
growth
M), which
were
(data
not shown).
mote
the formation
of feline
human
macrophage
colonies
similarly
inhibited
in the presence
Recombinant
human
IL-3 failed
of myeloid
colonies,
and
neither
M-CSF
(CFUof FPV
to proIL-3
nor
GM-CSF
would
support
the formation
of BFU-E
(data
not
shown).
In contrast
to myeloid
colony
formation,
erythroid
colony
formation
stimulated
by the presence
of burst promoting
activity
modestly
in FeLV-A-FEF
affected
by FPV.
conditioned
medium
was
only
± 1.4;
that
colony
in mitotic
of
29%
and
39%
may
±
result
the
forming
cells
experiments.
was:
CFU-E,
CFU-GM,
CFU-E
virus
because
greater
susceptibility
primitive
erythroid
addition
Recombinant
hematopoietic
GM-CSF)
CFU-G,
to
ences
to cultures.
of
27%
recombinant
GM-CSF);
both of which supported
the growth
of feline myeloid
colonies
(Fig
11). Myeboid
colony
formation
stimulated
by these
recombinant
growth
factors
was completely
inhibited
by the
of FPV
rates
virus
toxicity
was the
activity
of progenitors,
assessed
in 3H-thymidine
suicide
mabs,
inhibition
by thymidine
BFU-E,
contribureduced
serum.
To determine
whether
differences
in the mitotic
cycling
To
production
by accessory
by adherence
to plastic
erythrocytes;
of fetal
conditions,
addition
human
of virus
growth
factor
was depleted
heterobogous
CFU-E
CFU-GM
on specific
± 4.1
of
cell
was
In four ani71%
± 2.9;
(at
1,000
U/mL
22%
± 4.0
(10,000
U/mL
1.7 (x ± SEM).
These
data
be more
their
high
of myeloid
progenitors
susceptible
than
BFU-E
cycling
rate,
but that
the
progenitors
compared
with
was not the result
of differ-
activity.
DISCUSSION
results
implicate
progenitor
and
panleukopenia
FPV
in cats.
propagation
direct
precursor
infection
by FPV
of bone
cells in the pathogenesis
of
As a productive
in feline
bone
marrow
system,
the virus in feline embryonic
kidney
cells.34 Viral
was detected
after
inoculation
with
as little
plaque-forming
U/b6
U/20
to 50 progenitor
infectivity,
probably
reinfection
by newly
the kinetics
is similar
to that
propagation
as I to
nucleated
cells (or 1 plaque-forming
cells),
indicating
a very high rate
due to rapid
and multiple
rounds
synthesized
virus.
Southern
analysis
of
of
2
of
of
of
From www.bloodjournal.org by guest on February 3, 2015. For personal use only.
FPV
INFECTION
OF CAT
BONE
MARROW
IN VITRO
77
Fig 6.
In situ hybridization
of cat bone marrow
cells infected
with FPV in vitro. Cells infected
at a virus to cell ratio of 1 :4.00 were
harvested
at varying
times and cytocentrifuge
preparations
were subjected
to in situ hybridization
with either
a specific
S labeled cloned
FPV probe (pEH2O)
or a nonspecific
plasmic DNA (pUC19).
The experiment
presented
here was performed
under conditions
that detected
both single stranded
viral DNA and RNA. Less than 1 % of cells showed
grain deposition
at 24 hours (A). By 48 hours (B). 16% of cells were
positive
and by 72 hours (C). 25%. The number
of infected
cells subsequently
decreased
(approximately
10% by day 6). Negative
controls
included
infected
cells (48 hours) hybridized
with a labeled nonspecific
probe (pUC1 9; D) and uninfected
cells hybridized
with pEH2O (E). (F)
Productively
infected
feline embryo
fibroblasts
(FEF) hybridized
with labeled
pEH2O served
as positive
control.
Original
magnification
x215.
DNA
confirmed
first 24 hours
ruses
that replication
occurrred
postinfection),
as observed
Failure
progenitor
inhibition
larger
would
cell;
of
nascent
colony
indicate
that
in cultures
colony
number
FPV
virus
cannot
production
presumably,
of target
FPV
erythroid
genitors
virus
tent
occasional
cell
replication,
be detected,
propagation
infected
as
but it is not possible
early
Late
consistent
recognizable
was
myeboid
precurin infection
viral
with
a decline
granulocytic
is limited
to
in
precursors;
by the
availability
progenitor
appeared
colony
formation
cells,
but
to be
more
by both
by several
sensitive
First,
the relative
virus dose required
derived
colony
formation
compared
granulocytic
tests
myeloid
to virus
and
less and
was
formation,
two
but
curves.
CFU-GM,
possibly
or toxic
in some
in Fig 9, the
there
“hits”
have
rate
susceptibility
cally active
no difference
would
be consis-
by virus
on BFU-E
reflecting
differences
intracellular
of
human
and
colony
specific
factors
pro-
limited
recombinant
of cell
CFU-E
may
but
of cytotoxCFU-GM,
cycling,
the
contribute
was
similar
by
recombinant
M-CSF)
formation
requires
the
IL-3
growth
mitoti-
completely
and
able to
by the
factors
(G-CSF,
to our cultures.
In these
experistimulated
by these
myeloid-
experiments
lack
much
to their
perhaps
not unexpectedly,33’3
we were
the growth
of myeloid
progenitor
cells
GM-CSF,
ments,
to perform
rates
of
in
interactions,
to FPV, which as a parvovirus
cells for propagation.
Second,
stimulate
addition
similar
of cycling
for
compared
was
result
expericurve
right-shifted
This
for more
binding
CFU-G
and
inhibtion.
to inhibit
CFU-GMwith
BFU-E-derived
formation
the requirement
with
sixfold
As shown
it might
also be the result of distinct
mechanisms
icity in cells of these two lineages.
While
BFU-E,
higher
From these results,
of infected
progeni-
cells.
inhibited
with
by
after
the possibility
that
infected
by FPV.
and
of the
infected
when
visualized
hybridization
three days
methods
at 24 hours.
some of the progeny
observed.
colony
slopes
cell membrane
only
exclude
are directly
similar
burst
myeboid
in the
compared
by these
infer that
to viral
was
a
of
A
although
replication
less.
of
infection,
permissive
with
infects
cells,
calculated
determined
we would
rigorously
sor cells
about
proportion
the
to be
or in situ
in infected
directly
ofaccessory
by
than
an
formation
FPV
depleted
formation
of cells
progenitors
appeared
immunofluorescence
are
was
to 10 to 15-fold
inhibition
to see even
dishes
formation
ments
in cubtures.338
culture
tors
colony
early (within
the
for other
parvovi-
of
the
inhibited
by virus.
for the erythroid
species
absence
cross-reactivity
of feline
activity
in human
recombinant
GM-CSF.
conditioned
medium
rich in feline
burst
Attempts
lineage
of
burst
However,
promoting
were
human
promoting
when a
activity
From www.bloodjournal.org by guest on February 3, 2015. For personal use only.
KURTZMAN
78
ET AL
Fig 7.
Immunofluorescence.
Immunofluorescence
was performed
by incubation
of cytocentrifuge
perparations
of cultured
cat bone
marrow
cells wtih a specific
dog immune
serum (anti-CPV.
see Methods)
followed
by FITC-labeled
rabbit antidog IgG. Specific fluorescence
was detected
at 48 hours (A) and 72 hours (B) postinfection
predominantly
localized
to the nucleus.
(C) Infected
cells (72 hours) reacted
with the anti-CPV
antibody
followed
by FITC-labeled
rabbit antimouse
IgG was exposed
for the same time as in B and served as a negative
control.
(D) Infected
FEF cells reacted
with anti-CPV
followed
by FITC-Iabeled
rabbit antidog
lgG. Original
magnification
x 550.
was used
to stimulate
colony
formation,
FPV
burst
colony
cultures
also
FPV
formation.
inconsistently
propagation,
primitive
erythroid
efficient
at inhibiting
Erythopoietin
added
to suspension
and at best modestly
enhanced
in the
presence
of burst
promoting
recover.15
their
panleukopenia
die
Fig 8.
magnification
soon
after
Wright’s-Giemsa
x210.
is an acute
infection
stains
disease,
of
in which
septicemia
of infected
or
animals
rapidly
(A) and uninfected
Because
levels
in
production.
of
blood
the
are
Interruption
short
life-span
dependent
of
on
of
rapid
direct
(B) cat
bone
suppression
death
cytotoxicity
marrow
might
of the
of FPV
cells
not
animal
its
after
granulocytes,
marrow
even
a global
by
to affect
neutrophil
feline
panleukopenia,
be apparent
or
for marrow
harvested
of
continued
production,
marrow
infection,
would
be anticipated
levels
quickly.
Given
the course
of
erythroid
activity.
Feline
either
even
predominantly
was much
less
either
prompt
progenitors
six days
because
recovery.
also
in culture.
The
offers
Original
From www.bloodjournal.org by guest on February 3, 2015. For personal use only.
FPV
INFECTION
OF CAT
BONE
MARROW
79
IN VITRO
100
C
0
C’,
80
E
0
>‘
C
0
0
0
0
C
0
0
#{176}
U)
8,
.L’
E
.2
0
U
.
C
0
C.)
virus
-
Fig 1 0.
Antibody
neutralization
of FPV inhibition
of hematopoietic
colonies.
Virus
or virus
preincubated
with
an excess
of
neutralizing
antibody
was incubated
with bone marrow
mononuclear cells at a virus to cell ratio 1 :4 (which
had previously
been
shown to produce
maximal
inhibition
of colony formation).
Results
shown are the mean ( ± SEM) from two experiments.
Supernatant
from uninfected
NLFK cells (used to propagate
virus) showed
a
slight
stimulatory
effect
on colony
formation
in three
experiments.
CFU-E
I
..-..-.
viruses,
and
While
virus/progenitor
9.
Inhibition
of clonal
hematopoietic
colonies
by FPV.
Typical
experiment
(experiment
2. Table 1 ) in which
the response
of myeloid
(CFU-GM)
and early (BFU-E) and late (CFU-E)
erythroid
progenitor
cells to infection
with
varying
amounts
of FPV was
determined.
(Input virus is expressed
as the ratio of the number
of
infectious
virions
[pfu] to the number
of progenitor
cells per 1 x
10’ cells plated in control
uninfected
cultures).
The values plotted
represent
the mean
number
of colonies
on infected
duplicate
plates
as compared
with uninfected
control
cultures.
Error bars
represent
the standard
error of the mean.
One
possible
explanation
the
severity
of
experimental
host
cat’s
neutropenia
cent
infection
on primary
that
there
obtained
possibility
cells
finding
that
Bone
derived
from
degree
marrow
variability
in
natural
or
in the status
in these
of the
microbially
inno-
effect
of FPV
the cytopathic
litter
mates
was
of variability
from
different
litters
differences
in marrow
based on cell kinetics.42
These
studies
of FPV have obvious
ment experiments
with Bl9 parvovirus
row.38’43
either
be differences
a high
from
animals
of genetic
following
For example,
the mild degree
of
germ-free
animals
may be the result
proliferation
The
was
illness
may
marrow
animals.2’
panleukopenia.
for the extraordinary
clinical
bone marrow.
in infected
of reduced
of feline
cells
support
similiar
between
but
cells
suggests
the
susceptibility
in vitro
consistent
with
gation
propagation
of
both
are
cytotoxic
in vitro
has
to
progenitor
a high
on the addition
the absolute
on target
degree
cells.
of tropism
of specific
dependence
cell replication.
growth
factors,
of parvovirus
propa-
In outline,
both
viruses
show
similar
kinetics
of replication
in suspension
marrow
cultures,
distribution
of virus in a proportion
of marrow
cells, and
decline
in propagation
with
depletion
of a limited
target
cell population.
Long suspected
of a role in bone marrow
have resisted
detection.
The study
of their
hematopoietic
cells
and
probes.
molecular
system
of
requires
The
we describe
several
should
aspects
of
myeloid
cells,
and
viruses
with
the development
of immunologic
FPV-feline
marrow
bone
be amenable
marrow
to the
failure
the genetic
basis
a
progenitor
failure,
interaction
culture
investigation
caused
including
variables
affecting
susceptibility
role of the host immune
system,
the basis
by
viruses,
of stem cells,
for specificity
the
for
of virulence.
ACKNOWLEDGMENT
We
thank
William
providing
of
Drs
Hardy
Genetics
Banks
for their
feline
for
and
We
cells
and
growth
of the National
assistance
immune
Janis
like
Institute
with
and
Cornell
Drs
of
Peter
analysis
the
State
Drs David
of Arthritis
the statistical
Drs
and
and
Center
Steven
for
Clark
University
Tattersall
of Colorado
to thank
University
Cancer
serum,
Abkowitz
and
Carlson
also
of
Sloan Kettering
factors,
Jonathan
would
Carmichael
Memorial
Institute
Washington
plasmids.
Leland
from
us with
University
similarity
and complein human
bone mar-
viruses
cells, FPV shows relative
specificity
for myeloid
in both cases, virus infection
can be shown
to be
dependent
Fig
features
the
B I 9 parvovirus
for erythroid
progenitors;
100
for unusual
control
supematant
BFUE
.
----
an explanation
virus +
anti-CPVAb
CFU-GM
-
University
Alling
of
of
and
Infectious
of our data.
Yale
for
Steven
Diseases
From www.bloodjournal.org by guest on February 3, 2015. For personal use only.
Table
1 . Inhibition
of Cat Hematopoietic
CFU-GM
Experiment
No.
Control
Colony
BFU-E
moi,
moi
Control
1
34
±
3t
1.3
45
± 1
15.0
2
24
± 1
1.3
29
±
3
7.2
3
51
±
4
0.6
4
62
± 2
3.6
60
5
44
± 6
4.4
45 ± 1
23
± 1
Control
182
±
11
8.4
160
±
3.7
408
± 17
33.1
response
hematopoietic
was determined
progenitor
colony
tHematoroietic
by incubating
cell number.
formation
moi
varying
amounts
is the multiplicity
8
202
in comparison
with
of FPV with
of infection,
control
cultures,
bone marrow
calculated
calculated
BFU-E/CFU-GM
moi,
3.1
78 ± 3
Standard
Dose
by FPV
moiw
Geometric
to determine
Formation
CFU-E
11.7
colonies are expressed per 1 x i0 bone marrow mononuclear
2.4
0.8
5.4
0.6
0.3
14.3
0.4
7.0
1.0
2.0
2.7
7.6
0.6
5.9
0.9
1 .6
1.4
mean
error
mononuclear
as virus to progenitor
using
CFU-E/CFU-GM
mot,,,
cells, followed
cell ratio,
a Spearman-Karber
response
cells plated. Control represents
by plating
at which
there
in semisolid
was 50%
medium
inhibition
of
distribution.
mean (
± SD) of duplicate
uninfected
plates.
*The paired sample
§Paired sample t =
tfor ratio being different
-0.18
(P = 0.8)
100
from
one is 3.79
(P
=
.0 19).
A
20%
80
:::i
60
10%
5%
40
1%
Cl)
20
a)
C.)
‘1
0
CoC\o:*)(
x
.C\\
co:.*)
\
Cocso::)(
LC)
Cl)
a)
C
B
C
0
0
0
5%
80
11
10%
60
CFU-GM
CFU-G
::
30%
40
BFU-E
Jhli
20
t’
ctt
c,tt%
c,1’ ‘
.,\
-“‘x
Fig 1 1 .
FPV inhibition
of growth
factor
dependent
hematopoietic
colony formation.
Adherence
depleted
and rosetted
bone marrow
mononuclear
cells (see Methods)
were plated in semisolid
media at varying
concentrations
of FCS (to minimize
or eliminate
the effect of
undefined
growth
factors)
as indicated
above the bars. The effects
of (A) recombinant
human G-CSF (1 .000 U/mI)
and (B) (recombinant
human
GM-CSF
(10.000
U/mL)
on the formation
of granulocyte
(CFU-G).
mixed
myeloid
(CFU-GM).
and macrophage
(CFU-M)
colony
formation
was determined.
The controls
reprasent
the effect of FCS alone. Cells infected
with FPV ( + virus) were infected
at a virus to cell
ratio of 1 :400. (C) Effect of FPV on BFU-E. Adequate
burst formation
was only seen in the presence
of 30% FCS. CM represents
uninfected
cells grown
in the presence
of 7% conditioned
medium
from
FeLV-A
infected
FEF cells. a source
of cat burst promoting
activity.
FPV
infection
was performed
at a virus to cell ratio of 1 :400 (high) or 1 :4.000 (low). The control
is the number
of colonies
from uninfected
cells
in the presence
of 30% FCS alone. The results of typical experiments
shown here are presented
as the mean ( ± SD) of duplicate
plates.
From www.bloodjournal.org by guest on February 3, 2015. For personal use only.
FPV
INFECTION
OF CAT
BONE
MARROW
IN VITRO
81
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bone marrow
Bloodll:l448,
24.
virus
Hematologic
N EngI
marrow
in dogs:
E, Platanias
infection.
NS: Chronic
bone
1989
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NS:
and
1988
Leonard
Blood
Young
Viruses
G, Truman
109:695,
in aplastic
pathogenesis.
4.
2:51,
in the bone
Med
3. Young
phokines
NS:
B, Armstrong
Epstein-Barr
Ann
Young
K,
in vitro
Kurtzman
in human
Di,
with
Blood
panleucopenia
results.
G,
bone marrow
Moore
MAS,
recombinant
70:138a,
Souza
human
1987
virus.
IV.
Methods
Res Vet Sci 8:256,
Young
NS:
cell cultures.
LM:
granu-
Replicaiton
Science
for
1967
of
B19
233:883,
From www.bloodjournal.org by guest on February 3, 2015. For personal use only.
1989 74: 71-81
Feline parvovirus propagates in cat bone marrow cultures and inhibits
hematopoietic colony formation in vitro
GJ Kurtzman, L Platanias, L Lustig, N Frickhofen and NS Young
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