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Growth of rubella virus in cultures of synovial cells from rheumatoid arthritis.

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BRIEF COMMENT
Growth of Rubella Virus in Cultures of Synovial
Cells from Rheumatoid Arthritis
Lorne A. Rulnge and Anthony C. Allison
Nine rheumatoid and 5 control synovial cell cultures showed no deficiency in
ability to grow rubella virus or to exhibit a cytopathic effect. This finding considerably weakens previous evidence arguing for the presence of an agent within
rheumatoid synovial cells capable of producing intrinsic interference.
Symmetrical polyarthritis with synovial
effusions and morning stiffness usually signifies a process of undetermined etiology.
Some viral illnesses however, namely hepatitis, either Australia antigen positive (1) or
negative (2), and rubella (3) are capable of
producing a similar clinical syndrome.
This ability of viral infections to mimic
rheumatoid arthritis has encouraged the
search for a viral etiology. Interesting suggestive evidence includes interference with
viral replication of Newcastle disease virus (4) and rubella ( 5 ) in cells growing
from rheumatoid synovial explants. Because interference with rubella replication
was reported to be a feature of cells cultured from rheumatoid synovia, and to
differentiate these cells from control cells
( 5 ) , we decided to investigate this phenomenon further.
From the Clinical Research Centre, Harrow,
Middlesex HA1 SUJ, England.
LORNE A. RUNCE, MD: Fellow of the Canadian
Arthritis and Rheumatism Society; ANTHONY c. ALLISON, MB, PHD: Dircctor, Cell Pathology, Clinical Research Center, Harrow, Middlesex, England.
Address reprint requests to: Lorne A. Runge, MD.
Now located, Department of Medicine, Ottawa General Hospital, 43 Bruyere, Ottawa, Ontario, Canada.
Submitted for publication July 30, 1971; accepted
August 18, 1971.
MATERIAL AND METHODS
Synovia from patients with definite rheumatoid
arthritis were obtained during operative procedures
(5 knees, 3 wrists, 1 toe). Two samples came from
The Royal Hospital for Rheumatic Disease in Bath
and were transported in phosphate buffered saline
packed in ice, arriving in less than 6 hours. T h e
other rheumatoid and control synovia (3 menisectomy, 1 patellectomy [orteoarthritic], and 1 hip
replacement) were obatined here. T h e synovia were
minced and grown for the first few days in a small
amount of Eagle's basal medium modified for
diploid cells* supplemented with 25% calf serum in
a 5y0 CO, atmosphere on plastic petri dishes.?
Once the explants had stuck, the amount of
medium was increased and changed every 3-4 days.
Fibroblastic proliferation from the explants continued for 3-4 weeks, after which they were removed
with 0.257' trypsin in 1:5OOO versene, plated at
0.1-1.0 X 106 cells/ml in Eagle's basal medium
modified for diploid cells with 10% calf serum
(growth medium) in plastic culture flasks.?
Serial passages were performed when the monolayer became confluent (10-14 days). Five rheumatoid cultures reached a mean cell density of 6.5 X
lo6 cells per flask, and 5 controls were 1.02 X 108
cells per flask. Data illustrating the patients' ages at
the time of surgery and duration of synovial cell
culture prior to rubella testing are illustrated in
Figure 1.
Rubella virus strain GOS-10 was kindly supplied
by Dr. M. Clarke, National Institute for Medical
Research Laboratories, Hampstead. I t was isolated
from a child at Great Ormond Street Hospital, and
*Grand Island Biological Co, Grand Island, NY.
?Falcon Plastics, Oxnard, Calif.
Arthritis and Rheumatism, Vol. 15, No. 1 (January-February 1972)
85
RUNSE 81 ALLISON
SYNOVIAL
<4
CONTROL
51
2 4
44
I
I
MEANDURATION
INCULTURE (DAYS)
46.3
66.4
I
PATIENT
AGE NR)
1
I I
53. r
46.2
Fig 1. Characteristics of synovial cell cultures at time
of rubella infection.
passaged 7 times in RKlS cells and twice in BHK
cells. The titer fell from 4.2 X 1od TCID,/ml to
<lo* after incubation with commercial* human
antirubella complement-fixing antibody.
Recently confluent synovial cell monolayers in
60mm plastic petri dishes were infected with 1-3
TCID, units/cell in a volume of 0.3 ml. Adsorption
lasted 30 minutes at 20°C and 30 minutes at 33°C
with frequent agitation. The inoculum was removed, the monolayers washed and then
maintained in 5 ml of growth medium with
penicillin 100 units/ml and streptomycin 100 &g/ml
inside a sealed container gassed with 5y0 CO,, and
kept at 33°C. Samples were removed at various
intervals and stored at -7OOC until titrated. Medium was added when necessary to keep at least 3 ml
present per dish. Control samples were managed
similarly, using growth medium rather than rubella
virus.
TCID, assays were performed on microtest
tissue culture platest containing 1-2-day-old confluent RKlS monolayers grown in Eagle’s basal medium supplemented with 5y0 fetal bovine serum. 0.01
ml of 10-fold diluted samples in Eagle’s basal
medium and 2y0 fetal bovine serum were inoculated per well, 5 wells per dilution. Cytopathic
effects consisting of foci of granular degenerating
cells were observed at 1 week and TCID, levels
calculated. Reported values represent the average of
duplicate cultures or repeated measurements of the
same sample. All titrations and observations for
cytopathic effect were made without knowing the
sample source. Contaminated cultures were discarded.
RESULTS
T h e results of virus titration at various
time intervals for two separate experiments
are illustrated in Figure 2. No significant
difference is apparent, and as Figme 3
illustrates, the time at which the cytopathic
effect appeared is the same foi- rheumatoid
and control cultures. T h e cytopathic effect
involved increased cytoplasmic granularity,
rounding u p of cells, a decrease in cell
density, and a n increase in debris in the
medium. Uninfected monolayers consisterrtly remained normal in appearance.
Inoculum diluted with growth medium
without cells was titrated at various intervals, and no rubella could be demonstrated
from Day 4.5 onwards.
DISCUSSION
Interference with viral growth can occur
at many levels within the replicative cycle.
Problems with the host range of rubella is
probably not the basis for variable observations of growth in rheumatoid synovial
cells as control cells consistently grow the
virus. Otherwise sensitive cells can be made
resistant by interferon, or through the
presence of an interfering virus: intrinsic
interference (6). If deficient rubella replication were based on the latter phenomenon
-ie, a heretofore undesaibed rheumatoid
virus, one would expect failure of replication in spite of minor differences involving
culture conditions, strain of virus, titration
methods, etc. T h e fact that rheumatoid
synovial cells support growth of rubella in
some hands (5), and not others, indicates
that they probably are not exhibiting intrinsic interference. Unfortunately, this
variability of results considerably weakens
the evidence that an agent causing intrinsic
interference exists in all rheumatoid synovial cells.
ACKNOWLEDGMENT
+Wellcome Reagents Limited, Beckenham, England.
+Falcon Plastics, Oxnard, Calif.
86
We gratefully acknowledge the assistance of Drs.
M. Gumpel and D. Rubenstein in obtaining the
synovial tissue.
Arthritis and Rheumatism, Vol. 15, No. 1 (Januory-Februar)r 1972)
EXPERIMENT
TIME (DAYS)
1
2
Fig 2. Growth of rubella in synovial cells. The data is expressed as average TCI Dm units per culture
(duplicate or repeated).
a
3
A
AA
OIAA
AA
2
A
A 0
0
A
1
A 0
0
AAAOAA
c
U
P)
Y
0
.-
U
f
0
n
0
c
h
U
a
A
AAAAA
A0"
A
I
A00
I
I
I
I
1
I
I
I
I
I
I
I
I
I
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20
I
I
I
I
I
l i m e (doys)
Fig 3. Development of cytopathic effect in synovial cell cultures. 1+ cytopathic effect refers to just
25% of cells, 3+ to 25-75%, and 4+ to over
discernible effect, 2+ to cytopathic effect present in
75%. Open triangles (A) refer to rheumatoid cultures in experiment 1, closed triangles (A)to rheumatoid
cultures in experiment 2. Open circles (0)
refer to control cultures during experiment 1, and closed circles
( 0 )to control cultures in experiment 2.
<
RUNGE & ALLISON
REFERENCES
1. Alpert E, Corton RL, Schur PH: Arthritis
associated with hepatitis: complement component studies. Arthritis Rheum 13:303,
1970 (Abstr)
2. Fernandez R, McCarty PJ: T h e arthritis of
viral hepatitis. Ann Intern Med 74:207-211,
1971
3. Yanez JE, Thompson GR, Mikkelson WM
et al: Rubella arthritis. Ann Intern Med
64~772-777, 1966
88
4. Smith C, Hammerman, P: Resistance of
rheumatoid synovial cells to infection with
exogenous virus. Arthritis Rheum 11:842,
1968 (Abstr)
5. Grayzel AI, Beck C: Rubella infection of
synovial cells and ,the resistance of cells
derived from patients with rheumatoid arthritis. J Exp Med 131:367-373, 1970
6. Marcus PI, Carver DH: Intrinsic interference: A new type of viral interference. J
Virol 1:334-343, 1967
Arthritis and Rheumatism, Vol. 15, No. 1 (January-Februaq 1972)
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growth, arthritis, virus, culture, synovial, rubella, rheumatoid, cells
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