Download Non-permissiveness of synovial membrane cells to human parvovirus

Journal of General Virology (1992), 73, 1559-1562. Printed in Great Britain
1559
Non-permissiveness of synovial membrane cells to human parvovirus
B19 in vitro
N. P. H. Miki t and J. K. Chantler*
Division o f Medical Microbiology, University o f British Columbia, 2733 Heather Street, Vancouver,
British Columbia V 5 Z 1M9, Canada
The ability of cultured human synovial cells derived
from synovial membrane and cartilage to support the
replication of human parvovirus B 19 was assessed. No
viral D N A synthesis nor viral antigens were detected
suggesting that B 19 virus is not capable of replicating
in synovial cells. The significance of this finding in
relationship to the pathogenesis of parvovirus arthritis
is discussed.
Human parvovirus B19 is a small (20 to 25 nm), nonenveloped virus containing a linear ssDNA genome of
5-5 kb (Summers et al., 1983). Although B19 is an
autonomous parvovirus, it is extremely fastidious.
Replication is restricted to mitotically active cells and
has only been demonstrated in erythroid progenitor cells
from the bone marrow (Mortimer et al., 1983; Ozawa et
al., 1987; Srivastava & Lu, 1988) and foetal liver
(Yaegashi et al., 1989; Brown et al., 1991). Although
prenatal infection is associated with widespread viral
infection of a variety of tissues including liver, heart and
lung (Salimans et al., 1989), the virus is generally found in
the erythroid progenitor cells which are widely distributed in the developing foetus. Such prenatal infections
frequently cause spontaneous abortion or hydrops fetalis
(Brown, 1989; Anand et al., 1987).
Post-natal infection with human parvovirus is associated with several clinical manifestations including
erythema infectiosum (EI) or fifth disease (Anderson et
al., 1983), and a transient aplastic crisis in patients with
pre-existing haemolytic disease (Saarinen et al., 1986). E1
is a mild exanthematous disease in children but is
frequently associated with post-infectious polyarthralgia
in adults (Reid et al., 1985). This parvovirus-associated
arthropathy is very similar to the joint manifestations
found following rubella infection or immunization
(Editorial, 1985; Anderson et al., 1985) with which it is
commonly confused. It presents as a symmetrical
polyarthritis of sudden onset in peripheral joints and is
normally short-lived. As with rubella arthritis, joint
symptoms are more common in adults than in children,
and in women than in men (Ford et al., 1988). Moreover,
parvovirus arthropathy, like rubella-associated arthritis,
can persist for months or even years in certain
individuals (White et al., 1985; Reid et al., 1985) and has
been associated with rheumatoid arthritis in a few cases
(Cohen et al., 1986; Stierle et al., 1987; Simpson et al.,
1984). The acute joint symptoms induced by human
parvovirus have been suggested to be immune-complexmediated as they are usually coincident with the
appearance of specific antiviral antibodies in serum.
Whether infection of joint tissue occurs is not known as
the presence of viral antigen or D N A in joint tissue has
rarely been tested. However, human parvovirus has been
detected in the synovial fluid of a patient with B 19 virusassociated arthritis (Dijkmans et al., 1988).
In view of the close similarity in the joint symptoms
produced by human parvovirus and rubella virus and our
previous evidence that, in vitro, cells and tissue derived
from human foetal joints are highly permissive to rubella
virus (Miki & Chantler, 1992; Huppertz et al., 1991), we
examined the ability of B19 virus to replicate in
mitotically active synovial membrane and cartilage
(SMC) cultures.
Human parvovirus in the form of a high-titre patient's
serum was provided by Dr Peter Tattersall, Yale
University, New Haven, Conn., U.S.A., who also
supplied us with rabbit polyclonal antisera to the B19
virus non-structural and fusion proteins. Human foetal
SMC cells were prepared as described previously (Miki
& Chantler, 1992). Bone marrow (BM) cells from a
patient with chronic myelogenous leukaemia was obtained from Dr K. Humphries (Terry Fox Laboratory,
Vancouver, Canada). BM cells were cultured in Iscove's
modification of Dulbecco's MEM (DMEM) containing
20~o feotal bovine serum, 5~o agar, lymphocyte-conditioned medium plus 1.5 units/ml erythropoietin.
t Present address: Biomedical Research Centre, University of
British Columbia, 2222 Health Sciences Mall, Vancouver, British
Columbia V6T IZ3, Canada.
0001-0860© 1992SGM
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1560
Short communication
(a)
(b)
1
2
3
4
(c)
1
O
5
2
6
3
7
4
8
5
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Fig. 1. Detection of BI9 virus DNA in infected (lanes 1 and 2) or
mock-infected(lanes 3 and 4) SMC in comparisonwith infected (lanes
5 and 6) or mock-infected(lanes 7 and 8) BM cells. Lanes 1, 3, 5 and 7
represent cell-associatedDNA and lanes 2, 4, 6 and 8 show the result
for supernatant fluid. SMC cultures (a) were examined for B19 virus
DNA at (i) 0, (ii) 17 and (iii)96 h post-infection,BM cultures(b) at (i) 8,
(ii) 48 and (iii) 96 h post-infection.(c) M-11, a sourceof infectiousB19
virus was used as the sample. Tenfold dilutions of extracted DNA
served to determine the limit of detection and were equivalent to (lane
1) 5 x 105 to (lane 5) 5 x 101 genomes.
The ability of parvovirus B 19 to replicate in SMC cells
was determined by quantifying intracellular and supernatant virus D N A levels at three time-points postinfection. The BM cells infected in parallel were used as
a permissive system to provide a positive control. In
addition, attempts to detect viral antigen by immunoperoxidase staining were carried out.
In Fig. 1, the results of dot-blot analysis for viral D N A
are shown. For this experiment, SMC and BM cells were
infected at an m.o.i, of 10 to 20, and were incubated for
various periods up to 96 h post-infection. At the time of
harvest, both the culture medium and the cells were
examined for virus DNA. The medium was treated with
polyethylene glycol 3500 (to a final concentration of
10%) to precipitate supernatant virus which was then
pelleted by centrifugation at 7800g for 20 min. D N A
was extracted from the viral pellet and cells by a
modified Hirt procedure (Rosenthal et al., 1983). The
extracted D N A was washed in 70% ethanol, vacuumdried and dissolved in distilled water. The samples were
denatured by heating to 70 °C for 30 min in 250 mMNaOH. They were neutralized with an equal volume of
2 M-ammonium acetate, and applied by Hybond N nylon
(Amersham) using a 96-well dot-blot apparatus. After
air-drying the D N A was cross-linked to the membrane
by exposure to u.v. light (312 nm) for 5 min. The probe to
detect the B19 virus D N A consisted of a 700 nucleotide
fragment of B19 virus D N A cloned into plasmid
pAT153, obtained from Dr M. J. Anderson (University
College, London, U.K.). The viral insert was excised
with PstI and purified by electrophoresis in a 1.2%
agarose gel, followed by isolation using Gene Clean
(BioCan). The purified insert was labelled with digoxigenin l l - d U T P using the random priming method.
Prehybridization (2 h at 42 °C) and hybridization (18 h
at 42 °C) were carried out in 5 x SSC, 0.1% sodium
lauryl sarcosine, 0.02% SDS and 0.5% blocking reagent
(Boehringer Mannheim). Denatured labelled probe
(50 ng) was added to the hybridization buffer. Following
hybridization, the filters were washed and bound probe
was quantified with an indirect immunodetection system
using alkaline phosphatase and nitroblue tetrazolium,
and 5-bromo-4-chloro-3-indolylphosphate as substrates.
The results shown in Fig. 1 indicate that B19 virus
D N A replication in SMC could not be detected (lanes 1
and 2). A small amount of B19 virus D N A was seen in
the cell-associated sample (lane 2, i) at 1 h post-infection,
but this did not increase in subsequent samples taken at
later time-points. It is presumed therefore to represent
non-specific adsorption of viral particles to the cell
surface, or uptake of virus which is unable to replicate
and is degraded intracellularly. In contrast to the results
for SMC, B19 virus D N A replication was detected in the
permissive BM cells (lane 6). An increased amount of
B19 virus D N A was found at 48 and 96 h post-infection
relative to that found at 8 h post-infection. Low levels of
B19 virus D N A were also detected in the culture fluid of
these cells, equivalent to approximately 104 infectious
particles derived from 2 x 103 cells/culture. This
indicates that complete replication and release of B19
virus was occurring in BM cells although at very low
yield as has been described by others (Ozawa et al., 1986).
Attempts to detect B19 virus antigen in SMC cells
infected for 24 to 9 6 h were also carried out by
immunoperoxidase staining. Polyclonal antisera to
either B19 virus capsid or non-structural proteins,
provided by Dr P. Tattersall, were used as primary
antibodies for the reactions. No specific viral antigen
staining was obtained (data not shown), confirming the
lack of B 19 virus replication in SMC cells.
Our conclusion is that SMC cells, derived from normal
human foetal joint tissue, are non-permissive to the
replication of B19 virus. This contrasts with our results
for a number of other arthritogenic viruses which were
found to replicate in both cell and organ cultures of
human joint tissue (Huppertz et al., 1991; Miki &
Chantler, 1992). In particular, rubella virus, which is
similar to parvovirus B19 in its clinical association with
joint symptoms, was found to replicate to high titre in
SMC cells and to penetrate deeply into the synovial
membrane in organ culture (Huppertz et al., 1991 ; Miki
& Chantler, 1992). Mumps virus, adenovirus and
varicella-zoster virus also replicated to a limited extent in
SMC, whereas coxsackie B virus was found to be totally
restricted (Huppertz & Chantler, 1991; J. K. Chantler,
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N. P. H. Miki & H. I. Huppertz, unpublished results).
Ross River virus, a togavirus associated with epidemic
polyarthritis in Australia, has also been shown to
replicate in synovial cell cultures (Cunningham & Fraser,
1985a) although persistent infections which occur with
rubella virus were not established (Miki & Chantler,
1992; Cunningham & Fraser, 1985b).
These results indicate that local replication and
persistence of virus in synovial membrane cells may be
involved in the pathogenesis of arthritis induced by
certain viruses, particularly rubella virus and mumps
virus. However the complete restriction of replication in
joint tissue of parvovirus B19 reported here, and of
coxsackie B virus described previously (Huppertz et al.,
1991) suggests that immunopathological mechanisms
alone may be involved in the joint inflammation
observed in association with infection by these viruses.
Parvovirus B19 arthritis has been suggested to be
immune complex-mediated as the joint symptoms coincide with the appearance of antiviral antibody (White et
al., 1985; Editorial, 1985). This is largely speculative
however as analysis of synovial fluid for B19 virus
antigen or D N A has rarely been carried out. In one study
of 76 patients with rheumatoid or inflammatory arthritis,
six patients with evidence of recent BI9 virus infection
were identified (as determined by the presence of IgM
antibodies specific for the virus). However neither B19
virus antigen nor D N A was detected in their synovial
fluid (Cohen et al., 1986). In a contrasting study,
Dijkmans et al. (1988) found evidence for B19 virus
D N A in the inflammatory exudate of a 33-year-old
woman with acute B 19 virus-associated arthritis. Clearly
the role of B19 virus in joint tissue inflammation of the
majority of patients with parvovirus infection needs to be
further investigated. Moreover, detection of viral antigen or nucleic acid in synovial samples is not indicative
of viral replication in the tissue unless foci of infected
cells containing intracellular B 19 virus D N A and protein
are identified. In cases of chronic parvovirus arthropathy, it would seem likely therefore that viral persistence occurs in an extra-articular site and that the
prolonged symptoms are generated by immunopathological mechanisms (Anderson, 1987).
We are indebted to Dr David Owen and Sam Trepanier (Department
of Anatomic Pathology, University of British Columbia, Vancouver,
Canada) for the provision of human joint material. Excellent technical
assistance was provided by Georgia TaM. This research was supported
by operating grants from the Heighway Foundation and the Mary Pack
Fund.
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(Received 20 January 1992; Accepted 17 February 1992)
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