J. gen. Virol. (1968), z, 37-42
With ~ plate
Printed in Great Britain
37
Identification o f Two Possible Types of Virus Particle in
Rubella-infected Cells
By I. H. H O L M E S AND M A R G A R E T C. W A R K
Microbiology Department, University of Melbourne, Australia
AND I. J A C K AND J O C E L Y N G R U T Z N E R
Royal Children's Hospital, Parkville, N. 2, Victoria, Australia
(Accepted 19 July 1967)
SUMMARY
Cultures of RK 13 and BHK2I ceils infected with rubella virus were examined by electron microscopy when the cultures showed maximal cytopathic effects. Infected RK 13 cells contained crystalline inclusions (spacing
t9o X) as well as typical virus particles of total diameter 60o X, with a
dense 300 ~, core. Identical particles also occurred in infected BHK2I ceils,
but in these no crystals were observed. Neither crystals nor particles were
found in control cells. The particles did not resemble myxoviruses.
INTRODUCTION
Despite the great progress that has been made during the last few years in the
study of rubella virus, uncertainties have remained concerning its structure, and hence
its classification. On the basis of its size (estimated by filtration), its sensitivity to ether,
and the probability that it is an RNA virus, it has been assumed that it is a myxovirus, or more likely a paramyxovirus like measles. Norrby et al. (I963) demonstrated
particles compatible with this interpretation, but, as the authors themselves commented, they were not convincingly virus-like. In addition, they made the important
observation, later confirmed by Cusumano (I966), that the density of the infectious
particles in sucrose was only I.O7 g./cm. 3. This seemed too low for a paramyxovirus:
for instance the densities of measles and respiratory syncytial viruses are between
I-2Z and I'Z5 g./cm. 3 in CsC1 (Norrby et al. I964; Bloth & Norrby, I966; Coates,
Forsyth & Chanock, I966) and Cusumano noted that rubella appeared to behave like
Rauscher virus (O'Connor, Rauscher & Zeigel, 1964) in that denser components
appeared in preparations which had been previously centrifuged.
Very recently, Kim & Boatman (I967) reported annulate lamellae, areas of proliferation of smooth endoplasmic reticulum and 'crystal lattice structures' in LLCM K 2 cells infected with rubella virus. In these cells rubella is not cytopathogenic.
We chose to examine cultures of two types of cell in which rubella is cytopathogenic, namely the transformed rabbit kidney cell line RKI3, and the baby hamster
kidney line BHKzI. We confirm some of the findings of Kim & Boatman (I967),
but in addition the increased yields of rubella virus obtainable in our cell lines make
tentative identification of the actual virus particles possible.
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38
I. H. HOLMES AND OTHERS
METHODS
Viruses and cells. We selected strains of rubella virus which had been isolated and
grown solely in the particular cell line in which they were to be examined.The strain of
rubella studied in R K I 3 cells is designated RCI-I-657 and was isolated in R K I 3 cells
from the urine of a Io-day-old congenitally infected infant; its identity was confirmed
serologically. It was passaged solely in R K I 3 cells and used at its i2th and I7th
passages2
The strain studied in BHK21 cells is BAYLORR-I which was isolated by Rawls et al.
(I965) from thyroid tissue o f a congenitally infected infant and passaged twice in
B H K 2 I cells before freezing and despatch to Australia. It was examined during its
first and second passages in B H K 2 I cells in this country.
Cell monolayers in roller tubes were inoculated with virus at a multiplicity of
o.oi to I.O TCD 5o/cell, and were examined 6 to 9 days after inoculation when cytopathic effects were maximal.
Electron microscopy. The cells were removed from the tubes after treatment with
0"05% t r y p s i n + o ' o 2 % E D T A in calcium-and-magnesium-free phosphate-buffered
saline. After centrifugation to form pellets, the material was fixed overnight in Millonig
buffered 4 % (v/v) glutaraldehyde (Pease, I964), postfixed for 2 hr in 1% (w/v) osmium
tetroxide in the same buffer, dehydrated in ethanol and embedded in cross-linked
methacrylate (Kushida, 1961) or araldite (Glauert, 1961). Micrographs were taken at
magnifications of I o to 20,000 x using a Hitachi HU- I I A electron microscope operating
at 50 kv.
RESULTS
TWO per cent of sections of R K I 3 cells fixed 6 or 8 days after inoculation contained crystalline inclusions (P1. i, fig. I, 2). These were sometimes free in the cytoplasm, often in notably fibrillar areas. P1. i, fig. 2, shows two crystals from a group of
12 which were found in the remains of an almost completely lysed cell: note that these
are embedded in an unusual reticulum which is not typical of R K I 3 debris. Such
crystals were not found in control cells.
About 8 ~ of sections of R K 13 cells contained virus particles of the type illustrated
in P1. I, fig. 3. These had an electron-dense core of diameter about 3oo ~,, and a total
diameter of about 6oo ~,. They were found usually in small vesicles, frequently in the
region of the Golgi complex. Large numbers of similar particles were associated with
B H K 2 I cells 6 to 9 days after inoculation. Most particles were extracellular or in
phagocytic vesicles close to the cell surface, but some cells such as the one in P1. I,
fig. 4, contained structures which may be associated with virus synthesis. The cytoplasm
of this cell was filled with vesicles and tubules of endoplasmic reticulum and scattered
ribosomes, and the arrow indicates a particle apparently budding into a vesicle.
Unusually large numbers of particles surrounded this particular section and it seems
safe to infer that they had been released from this cell. No crystals were found in
B H K 2 I cultures.
We have not found any annulate lamellae or unusual membraneous inclusions such
as those described by Kim & Boatman (1967) in either of our cell lines.
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Rubella virus identification
39
DISCUSSION
The appearance of the 600 ~, particles and what we interpret as their sites of production suggest atfinities with mouse hepatitis virus whose multiplication cycle was
illustrated so excellently by David-Ferreira & Manaker (I965), and possibly with the
latent hamster virus observed by Bernhard & Tournier (I964) in hamster kidney cells
(including the BHK2t line). Both of these viruses, however, were reported to be
slightly larger (total diameters 700 and 850 ~ respectively).
We are faced with two most interesting possibilities. First, the 600 ~, particle may
indeed be the rubella virus. This type of particle would fulfil various characteristics
ascribed to rubella virus: it could be retained by a membrane filter of average pore
diameter 7Io ~, but would pass a 3ooo~, one (McCarthy, Taylor-Robinson &
Pillinger, i963). It could have a low density and be ether sensitive, since the mechanism of its extrusion into vacuoles by budding (P1. I, fig. 4) could be taken as evidence
for the presence of a lipid-containing outer membrane. Nevertheless, this virus does
not resemble a myxovirus or a paramyxovirus. It is substantially smaller than the
smallest known myxovirus; it has a well-defined electron-dense core with no indication
of helical substructure and its outer membrane has a relatively smooth outline without any suggestion of the projecting spikes characteristic of the myxovirus-paramyxovirus groups.
The second interesting alternative is raised by the presence of the crystalline inclusions. These must surely be related to the multiplication cycle of rubella, since they
were found by Kim & Boatman (I967) in LLC-MK2 cells and by us in R K I 3 cells,
the only common factor being inoculation with rubella. They could represent viral
protein produced in greater excess in some cell lines than in others. On the other hand,
they could actually be crystals of very small virus particles. Breese & Graves 0966)
have recently shown crystals of the well-characterized foot-and-mouth disease virus, a
small isometric RNA virus of diameter 230 ~,: these bear a striking resemblance to the
crystals shown here in P1. i, fig. i. It should be noted that in the case of close-packed
crystals of particles less than about 250 ~ in diameter, average sections (thickness say
400 ~,) will contain 2 or more layers of particles and since crystal planes will be cut at
random angles, various parallel spacings of width greater than the true particle spacing
may be observed. The minimal spacing observed is that arrowed in P1. I, fig. 2, where
the units are in a square array, centre to centre spacing about I9o ~,. If 'square"
spacings are measured, the crystals illustrated by Kim & Boatman (I967) give the
same result. Thus one must also consider the possibility that these are crystals of
rubella virions. An isometric virus of I9o/~ diameter would not be expected to have a
low density, but association with lipid-containing ceil debris could explain both filtration and density findings, as Cusumano (I966) suggested.
The 600 ~, virus could be a helper virus normally present in low concentration in
RK I3 and BHK2I cells and associated with rubella cytopathic effects in these cell
lines. The behaviour of rubella virus on first isolation in RK I3 cells does seem to vary
somewhat from one laboratory to another, and the interesting but puzzling results of
Butler (I966) showing that plaque-forming efficiency of rubella virus in R K I 3 cells
depended on the previous passage history of the strain could easily be explained
by a helper virus hypothesis. In ceils where a helper virus is necessary for multiplication, inactivation of either component virus could cause loss of'infectivity'. Some of the
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4o
I. H. HOLMES AND OTHERS
" e s t a b l i s h e d ' characteristics o f rubella virus w o u l d require revision i f this possibility
were confirmed.
I n c o l l a b o r a t i o n with D r M. F. W a r b u r t o n we are a t p r e s e n t e x a m i n i n g rubella
h a e m a g g l u t i n a t i o n using p i g e o n erythrocytes a n d h a e m a g g l u t i n i n f r o m R K 13 cells
infected w i t h rubella virus. A detailed r e p o r t o f o u r findings is in p r e p a r a t i o n , b u t it is
p e r t i n e n t to m e n t i o n here t h a t we have f o u n d 60o ~, particles like those in P1. I, fig. 3, 4
at the surface o f the a g g l u t i n a t e d erythrocytes, a n d since the h a e m a g g l u t i n a t i o n c a n
b e inhibited b y h u m a n convalescent rubella antisera, it is highly p r o b a b l e t h a t these
are the rubella virions.
W e w o u l d like to t h a n k the N a t i o n a l H e a l t h a n d M e d i c a l R e s e a r c h C o u n c i l o f
A u s t r a l i a for financial s u p p o r t , a n d M r T. L o for his efficient technical assistance.
W e especially t h a n k N o r e e n I. L e h m a n n o f Fairfield H o s p i t a l for s u p p l y i n g us with
infected a n d c o n t r o l cultures o f B H K 2I cells. W e are m o s t grateful to D r s K . K i m a n d
E. B o a t m a n for the o p p o r t u n i t y to s t u d y s o m e o f their p h o t o g r a p h s a n d findings
before p u b l i c a t i o n , a n d t o D r D. O. W h i t e f o r his m o s t helpful discussions a n d
comments.
REFERENCES
BERNHAm~,W. & TOUghER, P. (1964). Infection virale inapparente de eellules de hamsters d6cel6e par
la microscope 61ectronique. Annls Inst. Pasteur, Paris lO7, 447BLOTH, B. & NORRBY, E. (1966). Fractionation of respiratory syncytial (RS) virus components by
centrifugation techniques. Arch. ges. Virusforsch. x9, 385.
B~ESE, S. S., J t m . & GgAws, J. H. (1966). Electron microscopic observation of crystalline arrays of
foot-and-mouth disease virus. J. Bact. 92, I835.
BtrrLER, M. (I966). Rubella virus and the plaque test in RK 13 cultures. J. gen. Microbiol. 35, viii.
COATES,H. V., FORSYm, B. R. & CrIANOCK,R. M. (1966). Biophysical studies of respiratory syncytial
virus. I. Density of respiratory syncytial virus and associated complement-fixing antigens in a
caesium chloride density gradient. J. Bact. 9 x, I263.
CUSUMANO, C.L. (1966). Density gradient centrifugation studies of rubella virus. Prec. Soc. exp.
Biol. Med. xz2, 46I.
DAVrO-FERe,~mA,J. F. & MANnr,.ER,R. A. (1965). An electron microscope study of the development
of a mouse hepatitis virus in tissue culture cells. J. Cell Biol. 24, 57.
GLAUImT, A. M. (I96I). Techniques for Electron Microscopy. Ed. by D. Kay. Oxford: Blackwell.
K ~ , K. S. W. & BOATMAN,E. S. (I967). Electron microscopy of monkey kidney cell cultures infected
with rubella virus. J. ViroL x, 2o5.
KuslamA, H. 0961). A new embedding method for ultrathin sectioning using a methacrylate resin with
three dimensional polymer structure. J. Electron Microsc., Ciba Cy xo, 194.
McC~TaY, K., TAYLOR-ROBrNSON,C. H. & PmLIN6ER, S. E. (1963). Isolation of rubella virus from
cases in Britain. Lancet 2, 593.
Nol~RBY, E., MA6NUSSON,P., FRmIN6, B. & G ~ , S. 0963). A note on the morphology of rubella
virus. Arch. ges. Virusforsch. x3, 421.
N e R v y , E. C. J., MAGNOSSON,P., FALKSWDEN,L-G. & GRONBERa, M. 0964). Separation of measles
virus components by equilibrium eentrifugation in CsC1 gradients. II. Studies on the large and the
small haemagglutinin. Arch. ges. Virusforsch. x4, 462.
O'CONNOR, T. E., RAUSCHER,F. J. & ZEIGEL,R. F. (1964). Density gradient centrifugation of the
murine leukemia virus. Science, N.Y. x44, 1144.
PEASE,D. C. (I964). Histological Techniques for Electron Microscopy. New York, London: Academic
Press Inc.
RAVELS, W.E., MELr~CK, J.L., ROSE~CaER~, H.S. & BAYATPOUR, M. 0965)- Spontaneous virus
carrier cultures and post mortem isolation of virus from infants with congenital rubella. Prec. Soc.
exp. BioL Med. x2o, 623.
( R e c e i v e d I9 June 1967)
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Rubella virus identificatton
E X P L A N A T I O N OF PLATE
(See overleaf)
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4I
42
I. H. H O L M E S A N D O T H E R S
E X P L A N A T I O N OF P L A T E
The scale markers indicate zooo ~.
Fig. I. Crystalline inclusion in cytoplasm of a rubella-infected R K t 3 cell. Crystal is in a fibrillar area
adjacent to lipid droplets (L) and mitochondrion (M). Cross-linked methacrylate, lead citrate staining.
Fig. 2. Two crystals in reticular material among debris of a lysed R K t3 cell. M = mitochondrion,
Arrow indicates area of square packing in lattice. Cross-linked methacrylate, lead citrate.
Fig. 3. Virus particles (VP) within cytoplasmic vacuole in rubella-infected R K 13 cell. N M = nuclear
membrane. Cross-linked methacrylate, stained with uranyl acetate.
Fig. 4- Rubella-infected B H K 2i cell showing extracellular virus (VP) and probable budding of a
particle within a vesicle (arrow). Araldite, stained with tead citrate.
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Journal of General Virology, Vol. 2, No. x
I. H. HOLMES, M. C. WARK, I. JACK AND J. G R U T Z N E R
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Plate I
(Facing p. 42)