Download Enterovirus receptors and virus replication in human leukocytes

Journal
of General Virology (1999), 80, 921–927. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Enterovirus receptors and virus replication in human
leukocytes
Tytti Vuorinen,1 Raija Vainionpa$ a$ ,1 Jyrki Heino2 and Timo Hyypia$ 2, 3
1, 2
Department of Virology1 and MediCity Research Laboratory2, University of Turku, FIN-20520 Turku, Finland
3
Haartman Institute, Department of Virology, POB 21, University of Helsinki, FIN-00014 Helsinki, Finland
Although enteroviruses cause a great variety of diseases including meningitis, paralysis and
myocarditis, the life-cycle of these viruses in man is still quite poorly understood. The role of human
leukocytes as a target for enterovirus infections was studied in this report. Despite great similarity
in the structure and replication of coxsackievirus B3 (CBV3), echovirus 1 (EV1), and poliovirus 1
(PV1), the ability of these viruses to infect human peripheral blood mononuclear cells (PBMC), and
B (Raji), T (Molt-4) and monocytic (U-937) cell lines differed markedly. CBV3 attached both to
PBMC and the three haematopoietic cell lines studied, whereas EV1 bound only to PBMC.
Generally, the attachment of PV1 was very poor. The binding assays mostly correlated with the
expression of CD55 (decay accelerating factor, DAF) and α2β1-integrin (VLA-2), which are known to
act as receptors for CBV3 and EV1, respectively, as well as with the expression of the PV receptor
on the cell surface. To analyse virus replication in the cells, viral protein and nucleic acid syntheses
were studied by immunoprecipitation and RT–PCR. CBV3 was able to replicate in Raji and Molt-4
cells, even though no expression of DAF was detected, whereas in the monocytic cell line, viral
protein synthesis was detected only after transfection of virus RNA. Neither CBV3 nor EV1
replicated in PBMC and all haematopoietic cells studied seemed to be poorly permissive for PV1
replication.
Introduction
Enteroviruses (polio-, coxsackie-, echo- and enteroviruses
68–71) are small RNA viruses in the family Picornaviridae.
They are important human pathogens often causing mild
febrile illness, but clinical manifestations of enterovirus
infections also include meningitis, encephalitis, paralysis and
myocarditis. In infants, the course of the disease can be severe
due to multisystem involvement.
Primary replication of enteroviruses takes place in tissues of
the respiratory and gastrointestinal tracts. This may be
followed by the occurrence of infectious virus in blood and
lead to spreading of the pathogens to secondary target organs.
During this viraemic phase, enteroviruses have been cultured
from the serum of patients (Dagan et al., 1985 ; Prather et al.,
1984) indicating haematogenous spread of the viruses in free
form. The finding that enteroviruses can also be isolated from
blood cells of patients (Dagan et al., 1985 ; Prather et al., 1984)
Author for correspondence : Tytti Vuorinen.
Fax j358 2 2513303. e-mail tytti.vuorinen!utu.fi
0001-5887 # 1999 SGM
and the fact that some enteroviruses are able to replicate in
human peripheral blood mononuclear cells (PBMC) in vitro
(Dagan & Menegus, 1992 ; Freistadt et al., 1993) indicate that
infected cells may have a role in transporting the virus in the
body.
The initial step in virus infection is binding of the viruses to
specific receptors on the cell surface. In recent years, several
enterovirus receptors have been identified and characterized at
the molecular level. The poliovirus receptor (PVR) belongs to
the immunoglobulin superfamily. Although its structure has
been described in detail (Mendelsohn et al., 1989), its cellular
function is still unclear. Integrins serve as receptors for some
enteroviruses. Echovirus 1 (EV1) binds to α β -integrin (VLA# "
2 ; Bergelson et al., 1992, 1993), coxsackievirus A9 (CAV9) can
utilize αvβ -integrin in target cell recognition (Roivainen &
$
Hovi, 1989), while CAV21 and some other coxsackie A viruses
share a receptor (ICAM-1) with the major group of rhinoviruses (Colonno, 1987 ; Pulli et al., 1995). Decay accelerating
factor (DAF ; CD55) has been reported to be a receptor for
many echoviruses (Bergelson et al., 1994 ; Ward et al., 1994)
and it also plays a role in the attachment of coxsackievirus B1
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 08:10:42
JCB
T. Vuorinen and others
(CBV1), CBV3 and CBV5 to host cells (Bergelson et al., 1995 ;
Shafren et al., 1995). In addition, a molecule belonging to the
nucleolin family has been reported to recognize CBV3 on the
cell surface (Raab de Verdugo et al., 1995). More recently, a
common receptor molecule (CAR) for CBVs and adenoviruses
has been identified (Bergelson et al., 1997 ; Tomko et al., 1997).
Our previous results have shown that there are remarkable
differences in the susceptibility of haematopoietic cell lines to
CBV3 infection (Vuorinen et al., 1994, 1996). Both B and T cell
lines support virus replication, whereas monocytic and granulocytic cell lines are non-permissive for infection. In spite of the
susceptibility of B and T cell lines to CBV3 infection, we have
been unable to detect viral protein synthesis in in vitro-infected
PBMC originating from different donors (Vuorinen et al.,
1994).
In light of the previously published work, enterovirus
infection in leukocytes and haematopoietic cell lines is rather
obscure. One explanation for the variability of the results could
be differences in the expression of enterovirus receptors on the
surfaces of blood cells. Moreover, intracellular factors essential
for virus replication may play a role. In order to obtain a more
detailed picture on the interactions of enteroviruses with blood
cells, we have studied the attachment of radiolabelled CBV3,
EV1 and poliovirus 1 (PV1) to PBMC, as well as to monocytic,
B and T cell lines. Expression of the virus receptors on the cells
correlated with findings concerning viral macromolecule
synthesis in the infected cells.
Methods
Viruses, cells and cell lines. CBV3 (Nancy), EV1 (Farouk) and
PV1 (Mahoney) were obtained from the ATCC. Virus stocks were
propagated in LLC-Mk cells (ATCC), stored at k70 mC and centrifuged
#
at low speed prior to use in order to remove cell debris. Human PBMC
were isolated from healthy adults by Ficoll–Isopaque (Ficoll–Paque ;
Pharmacia) gradients. Haematopoietic cell lines (Raji, Molt-4 and U-937)
were originally obtained from the ATCC.
Infection and labelling of the cells, cell lines and viruses.
Cells (1i10' cells\ml) were infected at an m.o.i. of 5 in serum-free
Hanks’ balanced salt solution for 1 h at 37 mC, washed three times in
order to remove unabsorbed virus, and then maintained in RPMI-1640
medium supplemented with 10 % foetal calf serum (FCS) and 50 µg\ml
gentamicin. The cells for antigenomic RT–PCR were collected after the
adsorption as well as at 1, 2, 3 and 4 days post-infection (p.i.). The cells
were washed with PBS, 1 ml Ultraspec RNA (Biotecx Laboratories)
solution was added and the mixture was stored at k70 mC until total
RNA was isolated.
The labelling of the infected cells with [$&S]methionine was performed
at 3 h p.i. The cells were first cultured in methionine-free medium for 1 h
which was then replaced by medium containing [$&S]methionine
(50 µCi\ml ; Amersham). After 18 h, the cells were washed with PBS and
stored at k70 mC.
For labelling and purification of the viruses, infected LLC-Mk cells
#
were preincubated in methionine-free medium for 30 min and then
cultured in the medium containing [$&S]methionine. When complete
cytopathic effect was observed, the cultures were freeze–thawed three
times in order to release the intracellular virus. Cell debris was removed
JCC
by low-speed centrifugation, the virus was concentrated from the
supernatant by polyethylene glycol–NaCl precipitation and further
purified in linear 5–20 % sucrose gradients (Abraham & Colonno, 1984).
Isolation of RNA. Extraction of the RNA from the infected cells was
performed using the Ultraspec RNA isolation method with slight
modifications. In brief, 200 µl chloroform was added to the sample, which
was mixed, incubated on ice for 5 min and centrifuged at 12 000 g and
4 mC for 15 min. RNA was precipitated by isopropanol in the presence of
0n04 µg glycogen. The samples were stored at k20 mC overnight,
centrifuged at 12 000 g at 4 mC for 20 min, and the precipitates were
washed twice with 75 % ethanol. Finally, the pellets were dried at room
temperature, dissolved in RNase-free water, incubated at 55 mC for
15 min and stored at k70 mC until used in the RT reaction.
RT–PCR. An RT–PCR method was used to detect antigenomic
RNA. The oligonucleotide primers were from the conserved parts of the
5h-untranslated region of the enterovirus genome (Santti et al., 1997). The
cDNA reaction mixture (40 µl) contained 10 µl sample nucleic acid,
50 mM Tris–HCl (pH 8n3), 75 mM KCl, 3 mM MgCl , 10 mM DTT,
#
0n5 mM dNTPs (Pharmacia), 20 U RNasin ribonuclease inhibitor
(Promega), 50 pmol genomic (j) primer and 20 U murine Moloney
leukaemia virus reverse transcriptase (Promega) incubated at 37 mC for
1 h.
The PCR reaction (100 µl) consisted of 10 µl cDNA mixture, 10 mM
Tris–HCl (pH 8n8), 50 mM KCl, 1n5 mM MgCl , 0n1 % Triton X-100,
#
0n2 mM dNTPs, 50 pmol primers (Arola et al., 1996) and 1 U DNA
polymerase (Dynazyme). The mixture was amplified in a thermal cycler
(Perkin-Elmer Cetus) through 40 cycles of denaturation, annealing and
DNA synthesis for 1 min at 95 mC, 1 min at 55 mC and 4 min at 72 mC.
The reaction products (18 µl) were analysed in 2n5 % agarose gels
containing 1 µg\ml ethidium bromide.
Immunoprecipitation. The immunoprecipitation of viral capsid
proteins was performed according to a previously described procedure
(Vuorinen et al., 1994). The cells were lysed in 50 mM Tris–HCl (pH 7n4),
0n15 M NaCl, 1 mM EDTA, containing 1 % Tween and 1 % sodium
deoxycholate, sonicated and clarified by low-speed centrifugation. Rabbit
antiserum to purified CBV3 virions, rabbit antiserum to poliovirus and
horse antiserum to EV1 (ATCC) were used in the immunoprecipitation.
Protein A–Sepharose CL-4B beads and 20 µl of the antiserum were
incubated at 37 mC for 60 min. The labelled cell lysate was added, the
mixture was incubated overnight at 4 mC, washed four times with 0n1 M
Tris–HCl (pH 7n4), containing 0n01 % Tween, eluted with 0n1 M Tris–HCl
(pH 7n4), 2 % SDS, 5 % mercaptoethanol, and analysed by electrophoresis
in 12 % SDS–polyacrylamide gels. The dried gels were exposed on an Xray film.
Attachment of the viruses to the cells. Cells (1i10') were
washed once with Hanks’ balanced salt solution containing 10 mM
HEPES (pH 7n0) and 20 mM MgCl , incubated with 20 000 c.p.m. of
#
labelled virus in 0n1 ml medium at room temperature for 30 min. They
were washed three times with the medium, and the bound radioactivity
was measured in a liquid scintillation counter. Monoclonal antibodies to
DAF (IF7), α -integrin (12F1 ; Pischel et al., 1987) and PVR (D171) were
#
used to confirm the specificity of the binding assays.
Flow cytometric analysis. Cells (5i10&) were first incubated in
PBS containing 1 % FCS at 4 mC for 20 min, then with the anti-receptor
antibody for 30 min at 4 mC, and finally stained with FITC-conjugated
anti-mouse antibody (Zymed). Thereafter, the cells were washed twice
with PBS and 10 000 events were analysed by a FACScan cytometer
(Becton Dickinson). In order to quantify the expression of virus receptors
on the cells, mean fluorescence intensity (MFI) for each staining was
measured and the MFI ratio (MFIR) was calculated by dividing the MFI
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 08:10:42
Enterovirus replication in human leukocytes
of the staining with specific antibody by the MFI of the staining with
FITC-conjugated anti-mouse antibody only. MFIR 1n5 was considered
positive. Lymphocytes and monocytes were gated according to their
light scattering characteristics.
Transfection experiments. CBV3 RNA (1 µg) in 100 µl MEM growth medium (Gibco) was added to 10 µl Lipofectin reagent
(Gibco) premixed with 90 µl -MEM. After 10 min incubation period
at room temperature, 800 µl -MEM was added and washed cells
(1i10') were overlaid with the mixture. Fresh -MEM (2 ml) was
added after 2 h incubation at 37 mC and incubation was continued
overnight. The synthesis of viral polypeptides was then revealed by
immunoperoxidase staining (Waris et al., 1990) using rabbit antiserum
against heat-treated echovirus 11. The antiserum is known to be broadly
reactive among enteroviruses (M. Waris and others, unpublished results).
Results
Fig. 1 shows the flow cytometric histograms of the
expression of DAF (A), the α -integrin subunit (B) and PVR (C)
#
on the surface of the haematopoietic cell lines, human
lymphocytes and monocytes. LLC-Mk cells were included as
#
a positive control. Expression of DAF and the α -integrin
#
subunit was detected both on monocytes (MFIR of 1n5 and 4n7,
respectively) and the monocytic cell line U-937 (MFIR of 2n2
and 2n7), while PVR was detected on T (Molt-4) and monocytic
cell lines, as well as on monocytes (MFIR of 2n1, 6n5 and 2n0).
Cell number
Expression of the virus receptors on the cell surface
Attachment of the viruses to the cells
The attachment of CBV3, EV1 and PV1 to the haematopoietic cell lines and PBMC differed noticeably (Fig. 2). CBV3
bound to all the haematopoietic cell lines and to the peripheral
blood leukocytes of the five donors. The amount of attached
virus was highest on B (Raji) and monocytic (U937) cell lines
but a great variety occurred in attachment to the PBMC of the
five donors. EV1 had highest affinity to PBMC, whereas the
haematopoietic cells lines, including the receptor (α -integrin
#
subunit)-containing U-937 cells, did not bind the virus in
detectable amounts. PV1 had a very low capacity to attach to
all the haematopoietic cells studied. LLC-Mk cells, which are
#
known to be susceptible for the replication of these viruses,
were analysed as control cells.
Virus macromolecule synthesis in infected cells
Viral protein synthesis was analysed by immunoprecipitation using polyclonal antisera (Fig. 3). CBV3 polypeptides were detected in Raji cells (CBV3 ; lane 1) and weak
signals were also observed in the Molt-4 cells (CBV3 ; lane 2),
whereas the other cells did not support virus protein synthesis
in detectable amounts. PV1 proteins were synthesized in both
Molt-4 (PV1 ; lane 2) and U-937 (PV1 ; lane 3) cell lines. In
addition to the structural virus proteins, some other bands also
co-precipitated. The appearance of the co-precipitated polypeptides correlated to the susceptibility of the cells to virus
Relative fluorescence intensity
Fig. 1. Cell surface expression of DAF (A), α2-integrin (B) and PVR (C)
analysed by fluorescence cytometry. Unstained cells, cells incubated with
anti-mouse FITC–antibody alone, and cells incubated with DAF, α2-integrin
or PVR antibody before addition of FITC–antibody (shaded histograms) are
shown. The mean fluorescence intensity ratios are shown in the upper
right corner. Values 1n5 were considered positive.
replication suggesting that they may be virus-specific polyproteins. The interpretation of EV1 synthesis on the basis of
immunoprecipitation was not possible due to the high nonspecific background reactivity of the horse antiserum used.
Because the infectious virus contains positive-strand
genomic RNA, the synthesis of complementary (negative)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 08:10:42
JCD
T. Vuorinen and others
Fig. 2. Attachment of the [35S]methionine-labelled CBV3, EV1 and PV1 to
the haematopoietic cell lines and PBMC (hatched bars). The cells were
incubated at room temperature for 30 min with radioactively labelled virus
(" 20 000 c.p.m.) and the bound radioactivity was measured in a liquid
scintillation counter. The black bars show the attachment of the labelled
viruses to the cells after treatment with monoclonal antibodies against
DAF, α2-integrin or PVR antibody. MeanpSD c.p.m. is shown.
strands during the infection is a sensitive sign of de novo RNA
replication (Vuorinen et al., 1996). Therefore, we analysed the
amount of antigenomic RNA in the haematopoietic cells by
Fig. 4. Detection of antigenomic RNA by RT–PCR in B (Raji), T (Molt-4)
and monocytic (U-937) cell lines and PBMC infected with (A) CBV3, (B)
EV1 or (C) PV1 at either 1 h or 2 days post-infection. Lanes : C,
uninfected control cells ; MW, molecular mass markers. The arrows show
the position of the specific amplification product.
RT–PCR (Fig. 4), where the increase in the amount of
antigenomic RNA indicated productive RNA synthesis and
susceptibility of the cells to infection. Active CBV3 RNA
synthesis was observed in both Raji and Molt-4 cell lines but
not in U-937 cells or in PBMC (Fig. 4 A), whereas only U-937
Fig. 3. Synthesis of CBV3 and PV1
proteins detected by
immunoprecipitation. Cells were infected
and subsequently labelled with
[35S]methionine for 22 h. The cell lysates
were immunoprecipitated with CBV3 or
PV1 antibodies and analysed by
SDS–PAGE. Lanes : 1, infected Raji cell
line ; 2, infected Molt-4 cell line ; 3,
infected U-937 cell line ; 4, 5, infected
PBMC from two donors ; 6, infected LLCMk2 cells ; 7, uninfected LLC-Mk2 cells.
Virus polypeptides VP0, VP1, VP2 and
VP3 are indicated by the pointers.
Molecular mass markers are shown.
JCE
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 08:10:42
Enterovirus replication in human leukocytes
Table 1. Summary of results
Receptor expression*
Cell
Raji
Molt-4
U-937
PBMC
Virus binding
RNA synthesis
DAF
α2
PVR
CBV3
EV1
PV1
CBV3
EV1
PV1
k
k
j
j
k
k
j
j
k
j
j
j
j
j
j
j
k
j
k
j
k
k
j
j\k
j
j
k†
k
k
k
j
k
k
k
j
j
* A mean fluorescence intensity ratio (MFIR) 1n5 was considered to be positive.
† Viral protein synthesis was detected after transfection of genomic RNA.
used (data not shown). The unspecific staining was most
probably due to the known peroxidase activity of monocytes.
Discussion
Fig. 5. Viral protein synthesis after transfection of CBV3 genomic RNA into
the cells. The cells were stained by the immunoperoxidase method using a
broadly reacting echovirus 11 antibody. Raji cells were treated with
Lipofectin without (A) and with (B) CBV3 RNA. U-937 cells were
incubated with virus RNA in the absence (C) and presence (D) of
Lipofectin.
cells were able to support replication of EV1 RNA (Fig. 4 B).
PV1 RNA was produced in the U-937 cells and also in PBMC
(Fig. 4 C). Table 1 summarizes the results of receptor expression, virus binding and RNA synthesis.
To test whether the block in the CBV3 replication cycle in
the U-937 cells and PBMC is due to a lack of some intracellular
factor, we carried out transfection experiments with RNA
isolated from CBV3. Viral protein synthesis was clearly
detected by immunoperoxidase staining after transfection in
Raji and U-937 cells (Fig. 5). However, the interpretation of
immunoperoxidase staining of PBMC was not feasible because
unspecific staining of some cells was also detected even when
only growth medium, Lipofectin reagent or RNA alone was
Although enteroviruses have been isolated from blood cells
of patients (Dagan et al., 1985), detailed knowledge on the
interactions of enteroviruses with immune cells is still very
limited. Our previous studies have shown that B and T cell
lines support extensive CBV3 replication, but PBMC do not
produce significant amounts of virus (Vuorinen et al., 1994).
The purpose of the present study was to examine the blood cell
tropism of representatives of enteroviruses. The isolates
selected for study represent viruses with different molecular
and pathogenic characteristics. Polioviruses are the best known
members of the group and although they can cause epidemic
paralytic disease, they are more often responsible for asymptomatic and mild infections. CBVs are associated with diverse
clinical manifestations including meningitis, encephalitis and
myocarditis (Grist et al., 1978). Furthermore, increasing
evidence supports association of CBV infections with type 1
diabetes (Hyo$ ty et al., 1995). The largest enterovirus group is
the echoviruses, which comprises approximately 30 serologically distinct members. Echoviruses are a common cause of
meningoencephalitis and rashes.
In this study, PBMC were shown to contain specific
receptors (DAF, CD55) for CBV3, but neither viral macromolecule nor RNA synthesis could be detected in PBMC. Also,
the monocytic cell line U-937 expressed CD55 and virus
binding occurred, but no virus replication was detected by
RT–PCR or immunoblotting techniques. On the other hand,
immunoperoxidase staining experiments showed that transfected CBV3 RNA could be translated to proteins suggesting
incomplete uncoating or a defect in internalization during the
virus replication cycle. Recently, it has been shown that
interaction of CBV3 with two different receptor molecules,
DAF and CAR, is needed for initiation of infection (Shafren et
al., 1997). The expression of CAR on PBMC is currently
unknown. In spite of the absence of DAF on B (Raji) and T
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 08:10:42
JCF
T. Vuorinen and others
(Molt-4) cells, CBV3 was able to infect cells probably through
another receptor, possibly CAR, since synthesis of virus RNA
was detected in the cells. Our results with the monkey kidney
cell line LLC-Mk , commonly used for virus production,
#
showed that CBV3 binding can only partially be blocked by
the anti-DAF antibody, which also supports the idea of
alternative pathways for virus entry. Another explanation for
the finding could be that the monoclonal antibody produced
against human DAF is not equally effective in simian cells.
More efficient blocking was obtained in the blood cells and
CBV3 bound specifically to all three cell lines and to the
PBMC. However, significant variation was observed in the
binding to PBMC from different donors. In contrast to an
earlier report (Henke et al., 1992), we were unable to detect
signs of virus replication (RNA and protein synthesis) in
PBMC cultures from different donors ; thus, our results were
similar to the work reported by Dagan & Menegus (1992).
Our results showed that EV1 binds specifically to PBMC
and Molt-4 cells through the α -integrin subunit, whereas
#
virtually no binding to the Raji and U-937 cell lines could be
detected. Somewhat surprisingly, viral RNA synthesis was
observed in the U-937 cells that expressed the integrin on their
cell surface but were negative in the binding assay. However,
very small quantities of virus bound to the cell may also be able
to initiate infection. When replication of echoviruses 5, 9 and
11 has been previously studied, release of infectious virus was
clearly detected from blood mononuclear cells infected with
echovirus 5 and 11, but the virus titres from the cells infected
with echovirus 9 remained at a constant level during the
observation period (Dagan & Menegus, 1992). The molecular
basis of these differences is currently unknown. Recently, Ward
et al. (1998) showed that interaction between two receptor
molecules is needed for echovirus infection in RD cells. It is
possible that infection of haematopoietic cells is also a
multifarious phenomenon.
Polioviruses utilize PVR in their entry into the host cells
(Mendelsohn et al., 1989). We detected low levels of specific
binding of PV1 to the U-937 cells and to PBMC of at least one
donor. Virus replication was observed in the PBMC and U-937
cells although the quantity of RNA in the monocytic cell line
was low and could only be seen in the RT–PCR assay
detecting antigenomic RNA strands. The susceptibility of
PBMC to support growth of polioviruses appears to be
variable : Mastroeni et al. (1985) found only a very low level of
poliovirus replication while Freistadt et al. (1993) reported a
significant increase in the virus yield from infected PBMC
cultures.
Our data support the central role of the cellular receptors as
determinants of host cell tropism in enterovirus infections. In
CBV3 infections, more than one virus receptor evidently plays
a role. In the monocytic cell line U-937 lacking the specific
receptor, transfection of viral RNA into the cells was able to
initiate viral macromolecule synthesis, thus illustrating the
importance of early virus–cell interactions in the permissiveJCG
ness to infection. In general, there seem to be remarkable
differences when the three enteroviruses, using different
receptor systems for their entry into the blood cells, are
compared. Increasing knowledge on the molecular characteristics of the enteroviruses and further cell biological
investigations will make it possible to understand the complicated pathogenesis and clinical picture of enterovirus
infections.
We thank Jeffrey Bergelson for the DAF antibody, Eckard Wimmer
for the PVR antibody, Virgil Woods for the 12F1 antibody, Tapani Hovi
and Aimo Salmi for critical comments, and Marita Maaronen and Marjut
Sarjovaara for technical assistance. The study was supported by grants
from the Academy of Finland, the Sigrid Juselius Foundation and the
Turku University Foundation.
References
Abraham, G. & Colonno, R. J. (1984). Many rhinovirus serotypes share
the same cellular receptor. Journal of Virology 51, 340–345.
Arola, A., Santti, J., Ruuskanen, O., Halonen, P. & Hyypia$ , T. (1996).
Identification of enteroviruses in clinical specimens by a quantitative
polymerase chain reaction followed by genetic typing using sequence
analysis. Journal of Clinical Microbiology 34, 313–318.
Bergelson, J. M., Shepley, M. P., Chan, B. M., Hemler, M. E. & Finberg,
R. W. (1992). Identification of the integrin VLA-2 as a receptor for
echovirus 1. Science 255, 1718–1720.
Bergelson, J. M., Chan, B. M., Finberg, R. W. & Hemler, M. E. (1993).
The integrin VLA-2 binds echovirus 1 and extracellular matrix ligands by
different mechanisms. Journal of Clinical Investigation 92, 232–239.
Bergelson, J. M., Chan, M., Solomon, K. R., St John, N. F., Lin, H. &
Finberg, R. W. (1994). Decay-accelerating factor (CD55), a glycosyl-
phosphatidylinositol-anchored complement regulatory protein, is a
receptor for several echoviruses. Proceedings of the National Academy of
Sciences, USA 91, 6245–6249.
Bergelson, J. M., Mohanty, J. G., Crowell, R. L., St John, N. F., Lublin,
D. M. & Finberg, R. W. (1995). Coxsackievirus B3 adapted to growth in
RD cells binds to decay-accelerating factor (CD55). Journal of Virology 69,
1903–1906.
Bergelson, J. M., Cunningham, J. A., Droguett, G., Kurt-Jones, E. A.,
Krithivas, A., Hong, J. S., Horwitz, M. S., Crowell, R. L. & Finberg, R.
W. (1997). Isolation of a common receptor for Coxsackie B viruses and
adenoviruses 2 and 5. Science 275, 1320–1323.
Colonno, R. J. (1987). Cell surface receptors for picornaviruses. Bioessays
5, 270–274.
Dagan, R. & Menegus, M. A. (1992). Replication of enteroviruses in
human mononuclear cells. Israel Journal of Medical Science 28, 369–372.
Dagan, R., Jenista, J. A., Prather, S. L., Powell, K. R. & Menegus, M. A.
(1985). Viremia in hospitalized children with enterovirus infections.
Journal of Pediatrics 106, 397–401.
Freistadt, M. S., Fleit, H. B. & Wimmer, E. (1993). Poliovirus receptor
on human blood cells : a possible extraneural site of poliovirus replication.
Virology 195, 798–803.
Grist, N. R., Bell, E. J. & Assaad, F. (1978). Enteroviruses in human
disease. Progress in Medical Virology 24, 114–157.
Henke, A., Mohr, C., Sprenger, H., Graebner, C., Stelzner, A., Nain, M.
& Gemsa, D. (1992). Coxsackievirus B3-induced production of tumor
necrosis factor-alpha, IL-1 beta, and IL-6 in human monocytes. Journal of
Immunology 148, 2270–2277.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 08:10:42
Enterovirus replication in human leukocytes
Hyo$ ty, H., Hiltunen, M., Knip, M., Laakkonen, M., Vahasalo, P.,
Karjalainen, J., Koskela, P., Roivainen, M., Leinikki, P., Hovi, T.,
AH kerblom, H. K. & the Childhood Diabetes in Finland (DiMe) Study
Group (1995). A prospective study of the role of coxsackie B and other
enterovirus infections in the pathogenesis of IDDM. Diabetes 44,
652–657.
Mastroeni, P., Merendino, R. A., Bonina, L., Arena, A., Liberto, M. C. &
Gazzara, D. (1985). ‘ In vitro’ study on human mononuclear phagocytic
cells behaviour versus DNA and RNA viruses. Giornale di Batteriologia
Virologia ed Immunologia 78, 262–273.
Mendelsohn, C. L., Wimmer, E. & Racaniello, V. R. (1989). Cellular
receptor for poliovirus : molecular cloning, nucleotide sequence, and
expression of a new member of the immunoglobulin superfamily. Cell 56,
855–865.
Pischel, K. D., Hemler, M. E., Huang, C., Bluestein, H. G. & Woods,
V. J. (1987). Use of the monoclonal antibody 12F1 to characterize the
differentiation antigen VLA-2. Journal of Immunology 138, 226–233.
Prather, S. L., Dagan, R., Jenista, J. A. & Menegus, M. A. (1984). The
isolation of enteroviruses from blood : a comparison of four processing
methods. Journal of Medical Virology 14, 221–227.
Pulli, T., Koskimies, P. & Hyypia$ , T. (1995). Molecular comparison of
coxsackie A virus serotypes. Virology 212, 30–38.
Raab de Verdugo, U., Selinka, H. C., Huber, M., Kramer, B.,
Kellermann, J., Hofschneider, P. H. & Kandolf, R. (1995). Charac-
terization of a 100-kilodalton binding protein for the six serotypes of
coxsackie B viruses. Journal of Virology 69, 6751–6757.
Roivainen, M. & Hovi, T. (1989). Replication of poliovirus in human
mononuclear phagocyte cell lines is dependent on the stage of cell
differentiation. Journal of Medical Virology 27, 91–94.
Santti, J., Hyypia$ , T. & Halonen, P. (1997). Comparison of PCR primer
pairs in the detection of human rhinoviruses in nasopharyngeal aspirates.
Journal of Virological Methods 66, 139–147.
Shafren, D. R., Bates, R. C., Agrez, M. V., Herd, R. L., Burns, G. F. &
Barry, R. D. (1995). Coxsackieviruses B1, B3, and B5 use decay
accelerating factor as a receptor for cell attachment. Journal of Virology 69,
3873–3877.
Shafren, D. R., Williams, D. T. & Barry, R. (1997). A decay-accelerating
factor-binding strain of coxsackievirus B3 requires the coxsackievirusadenovirus receptor protein to mediate lytic infection of rhabdomyosarcoma cells. Journal of Virology 71, 9844–9848.
Tomko, R. P., Xu, R. & Philipson, L. (1997). HCAR and MCAR : the
human and mouse cellular receptors for group C adenoviruses and group
B coxsackieviruses. Proceedings of the National Academy of Sciences, USA
94, 3352–3356.
Vuorinen, T., Vainionpa$ a$ , R., Kettinen, H. & Hyypia$ , T. (1994).
Coxsackievirus B3 infection in human leukocytes and lymphoid cell lines.
Blood 84, 823–829.
Vuorinen, T., Vainionpa$ a$ , R., Vanharanta, R. & Hyypia$ , T. (1996).
Susceptibility of human bone marrow cells and hematopoietic cell lines to
coxsackievirus B3 infection. Journal of Virology 70, 9018–9023.
Ward, T., Pipkin, P. A., Clarkson, N. A., Stone, D. M., Minor, P. D. &
Almond, J. W. (1994). Decay-accelerating factor CD55 is identified as
the receptor for echovirus 7 using CELICS, a rapid immuno-focal cloning
method. EMBO Journal 13, 5070–5074.
Ward, T., Powell, R. M., Pipkin, P. A., Evans, D. J., Minor, P. D. &
Almond, J. W. (1998). Role for β2-microglobulin in echovirus infection
of rhabdomyosarcoma cells. Journal of Virology 72, 5360–5365.
Waris, M., Ziegler, T., Kivivirta, M. & Ruuskanen, O. (1990). Rapid
detection of respiratory syncytial virus and influenza A virus in cell
cultures by immunoperoxidase staining with monoclonal antibodies.
Journal of Clinical Microbiology 28, 1159–1162.
Received 7 August 1998 ; Accepted 11 December 1998
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 08:10:42
JCH