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Coxsackievirus B 3 Infection in Human Leukocytes and Lymphoid Cell Lines
By Tytti Vuorinen, Raija VainionpaB, Hilkka Kettinen, and Tim0 Hyypia
937 cell line with mononudearphagocyticcharacteristics
AlthoughCoxsackie B viruses (CBVs) are known to cause
was very limited. The virus was able to infect a small proporviremia during acute infection,the role of the blood cells as
tion of leukocytes and BM cells, and intracellular virus antia target for virus replication is poorly understood.We have
analyzed the susceptibility of human peripheral blood mono- gens were detected by immunofluorescent staining. Howof infectiousviruswas
ever,only adiminutiveamount
nuclearcells (PBMCS), granulocytes,bone marrow (BM)
produced in isolated PBMCs and granulocytes, and no virus
cells, and lymphoid cell linesto coxsackievirus B3 infection.
protein synthesis was detected by metabolic labeling and
Lymphoid cell lines with B- and T-cell characteristics (Raji
immunoprecipitationin these cells.
and Mot-4, respectively) supported virus replication
to high
titers and virus protein synthesis was detected by metabolic0 7994 by The Ameriwn Society of Hematology.
labeling and immunoprecipitation.CBV3 synthesis in the U-
C
OXSACKIE B viruses (CBVs), members of the enterovirus group in the Picornavirus family, are important
human pathogens and cause a great variety of diseases varying from the common cold to severe and occasionally fatal
infections, eg, myocarditis.’ After the initiation of enterovirus infection, the multiplication of the virus in the nasopharynx and in the alimentary tract is known to be often followed
by a short viremic phase when the virus can be isolated from
the blood. Despite the importance of this phenomenon in the
pathogenesis of infection (the blood stream can transfer the
virus to secondary target organs, eg, the heart) very little is
known about CBV infection in human blood cells.
However, it is known that different blood cell types harbor
enteroviruses in a different manner. Rather et a12 showed
that in 28 patients with coxsackie or other enterovirus infections, virus was isolated from blood in l1 cases and serum
was positive in 7, mononuclear leukocytes were positive in
9, and granulocytes were positive in 3 cases. Recently, it
was reported that coxsackievirus B3 is able to infect freshly
harvested human monocytes and to affect the immunologic
functions of the cells.3 On the other hand, Matteucci et a14
observed systemic lymphoid organ atrophy in coxsackievirus
B3-infected mice, but were unable to show virus replication
in thymus, spleen, and lymph nodes.
We report here that, although CBV3 antigens are found
in human peripheral blood mononuclear cells (PBMCs),
granulocytes, and bone marrow (BM) cells after in vitro
infection, the production of newly synthesized viral polypeptides or infectious virus was not detected. However, the
lymphoid cell lines with B- and T-cell characteristics are
highly permissive to CBV3 infection.
7.5% of monocytes. The granulocyte fraction contained 47% to 66%
granulocytes and 11% to 24% lymphocytes. Mononuclear cells were
cultured either unstimulated or stimulated with phytohemagglutinin
(PHA; 10 pg/mL, Difco Laboratories, Detroit, MI) 4 days before
infection. Human BM cells were aspirated from patients at the the
Department of Hematology, Turku University Central Hospital. BM
cells with morphologically normal appearance were used for virus
studies. Cells from at least 4 donors were tested in each experiment.
Raji, Molt-4, and U-937 cell lines were originally obtained from
ATCC.
Infection of cells. PBMCs, granulocytes, and BM cells as well
as Raji, Molt4 and U-937 cell lines were infected with the Nancy
strain of CBV3 at a low (1 or 5 ) and a high (100) multiplicity of
infection (m.0.i.) in 0.2 mL of serum-free Hanks’ balanced salt
solution. After the adsorption time of 1 hour, the cells were extensively washed to remove the unadsorbed virus inoculum. Infected
and control cells (lo6 celldml) were incubated at 37°C in RPM1
1640 medium, supplemented with 10% fetal calf serum (GIBCO
Europe, Glasgow, UK) and 50 pg/mL gentamicin. Lymphoblastoid
cell lines, PBMCs, and their supernatants were harvested for infectivity titration and spot hybridization test after different time intervals
postinfection ( p i ) during 96 hours, granulocytes were harvested
during a 24-hour incubation period, and both were stored at -70°C
until analyzed. The cells for the in situ hybridization and immunofluorescence (F)tests were collected after the adsorption time and
1, 2, 3, 4, and 5 days postinfection. Uninfected cells were used as
control material.
Infectivity titration. The release of CBV3 from infected cells to
the culture medium and the amount of intracellular virus was determined on confluent monolayers of LLC-MK, cell cultures in dilutions 10” to lo”* according to the appearance of a CPE. Before
inoculation, the cell suspensions were frozen and thawed three times
to release the virus and clarified by low-speed centrifugation. The
cells were examined for the CPE for 7 days and the results were
expressed as end-point titers.
MATERIALS AND METHODS
Preparation of virus stock. CBV3 (Nancy strain) was originally
obtained from American Type Culture Collection (ATCC; Rockville,
MD). The virus was propagated in roller cultures of LLC-MK, cells
(ATCC), which are Mycoplasma screened at regular intervals. When
cytopathic effect (CPE) of 75% to 100% was observed, the cells
and the culture medium were collected, frozen, and thawed three
times to release the virus, and stored at -70°C. Before use, the cell
debris was removed by low-speed centrifugation.
Cells and cell lines. Human PBMCs and granulocytes were isolated from healthy adults in Ficoll-Isopaque (Ficoll-Paque; Pharmacia Fine Chemicals, Uppsala, Sweden)/Histopaque-l119 (Sigma
Diagnostics, St Louis, MO) double gradients. According to the FAC
Scan flow cytometer analysis the mononuclear cell population of
five persons consisted of 84% to 94% of lymphocytes and 1.5% to
Blood, Vol 84, No 3 (August l ) , 1994: pp 823-829
From theDepartment of Virology and MediCity Research Laboratory, University of Turku, Finland.
Submitted February 3. 1993; accepted March 28, 1994.
Supported by grants from the Sigrid Juselius Foundation and the
Academy of Finland.
Address reprint requests to Tytti Vuorinen, MD, Department of
Virology, University of Turku, Kiinamyllynkatu 13, SF-20520 Turku,
Finland.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 V.S.C. section I734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-497I/94/8403-0006$3.00/0
823
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VUORINEN
824
AL
Fig 1. Coxsackievirus 63 RNA in infected Raji (A), Molt-4 (61, and U-937 (C) cell lines 24 hours postinfection detected by in situ hybridization.
D, E, and F represent corresponding dark fields. Original magnification x 25.
In situ hybridization. Ten to 20 pL of cell suspension was transfered onto polylysine-coated microscope slides and dried at room
temperature (RT). The cells werefixed in 4% phosphate-buffered
paraformaldehyde. Before hybridization, the slides were incubated
in0.2 mol/L HCI for 20 minutes; for 30 minutes in 2 X SSC at
70°C; proteinase K (1 mglmL)-treated for 15 minutes at 37°C in 20
mmol/L TRIS-HCI, pH 7.4, 2 mmol/L CaCI2;dehydrated in ethanol
series; and air dried. The slides were washed in aqua between the
treatments. The cDNA probe. covering most of the coxsackievirus
[email protected],
8 16 2LL8
oe...*
RAJ1
MOLT- 4
U-937
C
2
B3 genome, was labeled in a nick translationreactionusing "Sdeoxycytidine triphosphate ("'S-dCTP) and "S-deoxyguanosine triphosphate precursors to a specific activity of 1.5 X 1 Ox cpmlmL.
IO mmol/L
The hybridization solution contained 50% formamide,
TRIS-HCI, pH 7.4.600 mmol/L NaCI, I mmol/L EDTA. IO% dex0.1 mglmLrabbit
tran-sulfate,0.2 mg/mL salmonspermDNA,
tRNA, 0.1 mg/mL total Vero cell RNA, 0.05% bovine serum albumin, and 20 mmol/L dithiotreitol (DIT), and the denatured probe.
For each slide, 1.5 X I0"cpm of the probe was used, and the hybridization was performed at 25°C for
48 hours. Then the slides were
washed in 50% formamide, 600 mmol/L NaCI. I mmol/L EDTA,
I O mmol/L TRIS-HCI,pH 7.4, 20 mmol/L DTT, first briefly four
times, then twice for5 minutes at RT and overnight at 30°C. Finally,
the slides were washedfor 60 minutes at 55T, followed by two times
for 5 minutes at RT in 2 X SSC, 20 mmol/L DTT and dehydrated in
ethanol series containing 300mmol/L ammonium acetate. The slides
weredriedat42°Candcoated
for autoradiography with Nuclear
NY), diluted
Trackemulsion,NTB3(EastmanKodak,Rochester,
1:1 with 0.6 mol/L ammonium acetate, and exposed at 4°C for 14
days. After developing the film, the cells were counterstained with
hematoxylin and eosin. Plasmid vector pBR322
was used as a control
probe.
Spor hybridization. The proportional quantity of CBV3 RNA in
the infected cells was determined as described earlier by Vuorinen
et a16 using the following modifications: the CBV3 cDNAprobe'
waslabeled with "P-dCTPusing a nick translation kit (GIBCOBRL, Gaithersburg, MD) to a specific activity of 1.5 X IO' cpml
pg. After hybridization, the membrane was washed three times for
72 96 120 h.p.i.
4 6-8-.2L-h.p.i.
GRANULOCYTES
Fig 2. Detection of coxsackievirus 63-specific RNA by spot hybridization in infected Raji, MOM-4, and U-937 cell lines, and in isolated
granulocytes after different time intervals postinfection (h.p.i.). C
shows uninfected control cells.
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825
COXSACKIEVIRUS 63 INFECTION
infected with CBV3 at a high multiplicity of infection (MOI). After
3 hours p.i.,the cells were cultured for I hour in methionine-free
mediumandthen
labeled with S0 pCi/mL ”S-methionine. After
overnight incubation, lymphoid cell lines and PBMCs were washed
with phosphate-buffered saline (PBS) and stored at-70°C until
lysed in 0.05 mol/L TRIS-HCI. pH 7.4.0.15 m o l n NaCI, and 0.001
mol/L EDTA. containing I % Tween and I % sodium desoxycholate.
The lysates were sonicated for 1 minuteand centrifugated for 10
minutes at 8,OOO rpm to remove insoluble proteins. Protein A-Sepharose CL-4B beads and 20 pL of rabbit CBV3-antiserum were incubated at RT for 60 minutes. The labeled cell lysate was added and
the mixture was incubated overnight at 4°C. washed four times with
0. I m o l L TRIS-HCI containing 0.01 % Tween and eluted with 0. I
mol/L TRIS-HCI, 2% SDS, 5% mercaptoethanol. The immunoprecipitates were analyzed by electrophoresis in 12% SDS-polyacrylamide gels. The results were shown by autoradiography on an x-ray
film for 4 days andby using Phosphor Analyst (Bio-Rad Laboratories, Richmond, VA).
Inm,ltnr,fl~ore.~ce,Ice.Thecellswerefixed on slides in acetone at
until tested.Thefixedcells
4°Cfor 10 minutesandstoredat-20°C
were incubated for 45 minutes at 37°C with rabbit anti-CBV3 serum,
diluted 150 in PBS and further stained with fluorescein isothiocyanate
(F1TC)conjugated sheepantirabbit IgG (Dako, Glostrup,Denmark).
For the double-staining, cells werefirst incubated with the FITC-conjuJose, CA),
gatedanti-Leu-M3antibodies(BecktonDickinson,San
fixed in acetone at4°C.incubatedwithrabbitanti-CVB3serumand
finally stained with tetramethylrhodamine isothiocyanate-labeled
sheep
antirabbit IgG. The fluorescence was examined using a L e i t z Dialux
22 microscope (E. Leitz, Wetzlar, Germany).
-VPO
-vp3
A
B
C
D
E
F
Fig 3. Synthesis of virus proteins detected by immunoprecipitation with rabbit antiCVB3 serum. Uninfected Raji cell line (A), CBV3infected Raji cell line IBI, infected Molt-4 cell line (C), infected U 937
cell line (D), infected LLC-MK2 cells(E), and uninfected LLC-MK2 cells
(FI. Virus polypeptides VPO, VP1,VP2, and VP3 are indicated by
arrows. Molecular-weight markers 46, 30, 21.5, and 14.3 kD are depicted by lines on the left.
5 minutes at RT in 2 X SSC containing 0.I% sodium dodecyl sulfate
(SDS) and three times for 30 minutes at42°C in 0.1 X SSC containing 0. I ?+ SDS.
Immloloprec.ipirucIrioll. Raji, Molt-4,andU-937 cell lines,LLCMK? cells, as well as stimulated and unstimulated PBMCs were
RESULTS
Lymphoblnstoid cell lines. The in situ hybridization test
was used to show the proportion of cells containing viral
PBMC lM.O I
O-7
L
E
210
2L
L0
72
2L8
2L
L0 h.p.i 72
96hp1
c
a,
c
c
MOLT-L1M01.
.-C
0
Q
U
C
W
~O-~F
;lmuIate;
L
2 L 8 16 2L
10-5_
L0
72
U-937 lM.O I
PBMC ~ M . o . I .
~
~
O
-
~
~ IM.OT I E
96h.pl
210
10-5-
2Lh.p1
2~
210
L96h.pl
a
72
PHA stlmulated PBMC 100M.O. I
I
2L8
21
I
La h.p.i 72
L
”
2 L2 h6 p0 1
Fig 4. Production of infectious coxsackievirus B3 in the Raji, MOk-4, and U-937 cell lines, in unstimulated and stimulated PBMCs and in
granulocytes. Mean and SD of end-point titers from cells ( 0 )and the culture medium (0)
are shown. For PBMCs and granulocytes, results
with m.0.i. of 1 and 100 are shown.
S
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826
VUORINEN ET AL
Fig 5. Coxsackievirus B3 RNA in infected PBMCs (A), granulocytes (B),and BM cells (C) 24 hours postinfection detected by insitu hybridization. D, E, and F represent corresponding dark fields. Original magnification x 25.
RNA after the adsorption time of the virus and 24 hours p.i.
(Fig 1). Uninfected cell lines were used as controls. The
Molt-4 and Raji cell lines were highly permissive to CBV3
infection, whereas the monocytic cell line U-937didnot
show any positive signal. Viral RNA synthesis in the cell
cultures was also followed by analyzing the total quantity
of CBV3 RNA by spot hybridization (Fig 2). Also in this
assay, the Raji and Molt-4 cell lines were observed to support
virus RNA synthesis, whereas the monocytic cell line U-937
did not.
The synthesis of virus polypeptides VPO, VP1, VP2, and
VP3 was detected by immunoprecipitation from Rajiand
Molt-4 cell lines after metabolic labeling, but no virus-specific pro::ins were detected in the U-937 cell line (Fig 3).
The human lymphoblastoid cell lines with both B- and Tcell characteristics also supported growth of infectious virus
to high titers (Fig 4). and the replication cycle was similar
to that observed in standard cell cultures used to propagate
thevirus. The production of the virus started to increase
during the first 4 hours p.i. On the other hand, CBV3 production in the U-937 cell line was very limited or negative (Fig
4).
PB leukocytes and BM cells. By the in situ hybridization
test (Fig 5). only individual positive cells (=1/1,000) could
be detected in cultures of CBV3-infected PBMC and BM
cells, and in the spot hybridization test, the signals were
under the detection level (data not shown). However, immunofluorescent staining showed the presence of CBV3-antigen-positive cells already after the adsorption time. The
proportional number of the positive cells was between 10%
and 20%. The number of CBV3 positive leukocytes, both in
unstimulated and PHA-stimulated cells remained at a similar
level (Fig 6). The same proportion of the cells was positive
when lowor high m.0.i. was used. Althoughindividual variation (5% to 25% of positive cells) was observed, when
CBV3-infected mononuclear cell cultures of six donors representing different HLA-types were examined, comparable
results were obtained. Double staining showedthat some
cells of the monocyte population contained virus antigens
(Fig 6).
No release of infectious virus from PBMC infected with
either a low or high m.0.i. wasdetected, and PHAstimulation
had no effect on virus production (Fig 4). When 1 0 0 m.0.i.
was used, the end-point titers were at a higher level, obviously because of the high concentration of theinoculum
virus. No synthesis of virus polypeptides was detected in
infected unstimulated or stimulated PBMCs after metabolic
labeling and immunoprecipitation (not shown).
By IF test, CBV3 positive granulocytes were detected
already after 1 hour p i . (Fig 6). and after 24 hours p i ,
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COXSACKIEVIRUS B3 INFECTION
827
Fig 6. IF staining of CBV3 proteins in blood cells. Results obtained after the l-hour adsorption and 24-hour culture period are shown. A
and B show PBMCs infected with low rn.0.i. C and D show PBMCs infected with high multiplicity (100 m.o.i.1. G and H show PHA-stimulated
PBMCs infected with 100 MOL E and F show granulocytes infected with 1 rn.0.i. I and J show BM cells infected with 100 m.0.i. K and L show
double staining of PBMCs with antimonocytic antibody (Leu-M3; K) and anti-CVB3 (L).
the IF and the in situ hybridization tests (Fig S) showed a
proportional number (1/1,000) of CBV3 positive granulocytes. The signals in the spot hybridization test (Fig 2) were
weak, obviously detecting only RNA originating from the
adsorbed particles. No release of infectious virus to the culture medium was observed (Fig 4).
DISCUSSION
Viremia is associated with infections caused by a great variety of viruses. In some cases, eg, parvovirus B 19' and human
immunodeficiency virus'.' infections, it isknowntobe
caused by replication of the virus in defined subgroups of
blood cells. Although the target cells responsible for virus
replication have not yet been definitely identified, viremia is
also common in enterovirus infections.
Our earlier studies using a mouse model for coxsackievirus B3 infection showed that extensive viremia occurs during the first days after infection.' However, our attempts to
show viral infection of mouse leukocytes were unsuccessful,
indicating that the replication may rather take place in local
lymphnodes or in secondary target organs. Indeed,the
course of the viremic phase correlates well with the destruction of the exocrine pancreas, which could be the source of
the virus detected in the blood. However, in human infections, the destruction of thetarget organs isusuallyless
extensive, and therefore, other explanations for viremia are
needed.
CBV3 infection in humanPBMCswas observed to be
highly restricted. No distinct release of infectious virus was
detected in unstimulated or PHA-stimulated PBMCs (Fig 4).
This is different from other infectious viruses, eg, measles
virus, where PHA-stimulation extensively increases the synthesis of infectious virus."' The release of infectious CBV3
fromthe granulocytes was also minimalandthe
small
amount of virus detected during the first 24 hours incubation
time may originate from the inoculum. The relativelylow
proportion of virus-containing PBMCs can be explained by
infection of a subpopulation expressing the virus receptor.
It is also evident that the virusenters some cells by phagocytosis or related mechanisms. but does not replicate. Although
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VUORINEN ET AL
828
Henke et a13 observed CBV3 production in infected human
monocytes, we have been unable to detect active virus replication in these cells.
The IF test also showed CBV3 positive polymorphonuclear cells (Fig 6). It is not known whether these cells have
receptors for CBVs or if the viruses are taken into the cells by
phagocytosis. For instance, members of another enterovirus
group, echoviruses, have been found to change cell membrane structure of granulocytes and alter their adhesion properties.”.” It is possible that infected granulocytes or monocytes may transfer viruses through the circulation to different
target organs.
Virus infections are known to cause BM failure and the
role of hematopoetic cells as a target of virus infections has
arisen interest. Parvovirus is able to cause aplastic crisis in
patients with hemolytic anemia” and the virus is able to
infect precursors of mature erythrocytes in vitro.I4 Dengue
virus replicates in BM mononuclear cells in vitro,l5 whereas
cytomegalovirus infects stromal cells.16 We observed that
human BM cells were infected in vitro by CBV3, but the
number of infected cells was low, suggesting that BM does
not represent any main target for the replication of the coxsackieviruses.
When replication characteristics are concerned, CBV3 differs from polioviruses, which infect Molt4 and U-937 cells,
whereas no virus production is observed in Raji cell^."^^^
An important determinant in susceptibility to viral infection
is the cell surface receptor. Some of these molecules have
already been identified and it is known that
the poliovirus
receptor is a member of the Ig superfamily that is expressed
in many cell types.” Although the CBV receptor molecule
has been partially characterized, its exact nature is not known
to date? The variability inthe susceptibility ofhuman
lymphoid cell lines to these viruses could, at least partly, be
explained by the presence of different functionally active
receptors onthecell surface. However, other factors may
also play an important role because expression of the human
poliovirus receptor gene in the developing T-lymphocytes
in the thymus of transgenic mice is not sufficient for making
these cells susceptible to infection.”
We show here that a small proportion of in vitro infected
human PBMCs absorb CBV3 antigens, and granulocytes are
also able to harbor virus. According to the double-staining
IF, the PBMCs finding can be partly explained by the presence of the viral material in a monocyte population. Production of virus polypeptides in PBMCs was not observed by
immunoprecipitation even though a high concentration of
inoculunl virus was used and no clearly detectable synthesis
of infectious virus in the cultured cells occurred. Therefore,
the transient viremia cannot be explained by the replication
of thevirusin
these cells. However, it is evident that
absorptiodphagocytosis of CVB3 to the blood cells takes
place and it is possible that a small population supports
active virus replication. The results are unchanged when very
high virus concentrations are used. Further studies using new
approaches will be needed to shed light on the possible role
oflymph nodes and vascular epithelium as the origin of
enteroviruses found in blood during the viremic phase of
infection.
ACKNOWLEDGMENT
We thank Marjut Sarjovaara and Marita Maaronen for technical
assistance, Taina Kivela for secreterial help, Dr Reinhard Kandolf
for providing us with the CBV3 cDNA probe, and Dr Tarja-Terttu
Pelliniemi for the examination of the BM aspirates.
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COXSACKIEVRUS B3 INFECTION
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1994 84: 823-829
Coxsackievirus B3 infection in human leukocytes and lymphoid cell
lines
T Vuorinen, R Vainionpaa, H Kettinen and T Hyypia
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