/. Embryol. exp. Morph. Vol. 20, 3, pp. 285-94, November 1968
With 1 plate
Printed in Great Britain
285
The effect of
rubella and herpesvirus hominis on the pre- and
post-implantation stages of pregnancy in
laboratory animals
By J. S. OXFORD 1 & R. N. P. SUTTON 2
University of Sheffield, Virus Research Laboratory
The teratogenic effect of rubella virus in humans has now been well documented (Ingalls, Plotkin, Meyer & Parkman, 1967) and virus isolation studies
have suggested that rubella virus causes embryopathy by actually infecting the
cells of the developing foetus (Alford, Neva & Weller, 1964). However, a
laboratory animal model is required for controlled studies of the teratogenic
activity of rubella virus and for the assessment of possible preventative measures.
Such an animal system would also be useful for screening attenuated rubella
vaccine strains for teratogenic properties. Pregnant primates have been used in
preliminary experiments with rubella virus (Parkman, Phillips & Meyer, 1965)
but suffer from the disadvantages of size, expense and difficulty of mating. Both
the hamster (Ferm & Kilham, 1965) and the rabbit (Gibson, Staples & Newberne,
1966) have proved useful animals for the investigation of teratogenic agents. We
have described previously the adaptation of strains of rubella virus to small
laboratory animals (Oxford & Schild, 1966; Oxford, 1967) and wish now to
report studies of the infection of rabbits and hamsters with rubella virus in the
early stages of pregnancy.
It was considered necessary during the development of such an animal model
of teratogenicity to control the possibility that any embryopathic effect could
be caused indirectly by passage of toxic metabolites rather than by passage of
infective virus (Coffey & Jessop, 1959). Therefore the pre-implantation rabbit
blastocyst (Lutwak-Mann & Hay, 1962) was used in the present study to detect
any toxic activity. The rabbit blastocyst at this stage has been shown to be very
sensitive to the toxic action of a number of agents (Lutwak-Mann & Hay, 1962).
In addition, it is unlikely that any infective virus particles could reach the blastocyst before implantation (Zimmermann, Gottschewski, Flamm & Kunz, 1963).
In contrast, the post-implantation foetal stages of both the hamster and the
1
Author's address: University of Sheffield Virus Research Laboratory, Lodge Moor
Hospital, Sheffield, 10, England.
2
Author's address: Institute of Child Health, Great Ormond Street, London, England.
19
JEEM 29
286
J. S. OXFORD & R. N. P. SUTTON
rabbit were used as models to investigate the effects of any transplacental infection of the foetus with rubella virus. As controls for these experiments pregnant
animals were inoculated with teratogenic drugs such as trypan blue, triethylenemelamine (Adams, Hay & Lutwak-Mann, 1961) and also herpesvirus hominis,
which has been shown to cross the placenta of infected rabbits (Beigeleissen &
Scott, 1958).
MATERIALS AND METHODS
Virus strains
Rubella
The 'Judith' strain (supplied by Professor K. McCarthy, Liverpool) had been
passed twice in grivet monkey kidney cells and 40 times in a continuous line
of rabbit kidney cells (RK-13). The 'Porterfield' strain, recovered from a human
foetus, had been subsequently passed 15 times in RK-13 cells (Kay et al. 1964).
Herpesvirus hominis
A stock strain was supplied by Dr A. D. Macrae, Colindale, and passed 3
times on RK-13 cells and twice on baby hamster kidney (BHK-21) cells.
Cell cultures
RK-13 cells were used in attempts to recover infective rubella and herpesvirus
from foetal tissues. RK-13 cells were cultured in 'medium 199' containing 5 %
calf serum and 0-88 g/1. sodium bicarbonate and were maintained in the same
medium containing 2 % calf serum.
Experimental animals
Rabbits
New Zealand white does with a record of having raised three previous litters
with at least six offspring in each were purchased. Two males were used for all
mating experiments. Mating was observed and this considered as day 0 of
pregnancy.
Hamsters
Groups of eight female hamsters (Mesocricetus auratus) from a randomly
bred colony were caged with four males for 12 h overnight. The males were then
removed and the female hamsters caged individually.
Examination offoetuses for abnormalities
In the majority of the experiments with pregnant hamsters and rabbits the
foetuses were removed surgically shortly before term. This prevented the mother
eating any dead or deformed new-born. Each foetus was examined using a
dissecting microscope for abnormalities of the palate, eyes, ears, limbs and digits,
Rubella teratogenesis
287
axial skeleton and head. The weight and length of the foetus and any superficial
haemorrhages or oedema were also recorded. A proportion of foetuses were
X-rayed to observe bone structure, and others were serially sectioned. In some
experiments the mothers were allowed to give birth normally and the subsequent
development of the offspring was followed over a 5-month period. These animals
were examined for eye abnormalities, and hearing was tested by noting the
reaction to sudden high noise levels.
Attempts to recover viruses from animal tissues
Both maternal and foetal organs were removed aseptically with separate instruments for each organ and washed in three changes of phosphate buffered
saline (pH 7-2) to remove any superficial blood or contaminating virus. The
organs were minced in an M.S.E. blender and suspended in 'medium 199' +
2 % calf serum to 10 % (w/v). The tissue suspensions were centrifuged (800 g
for 10 min) and the supernatant stored at — 80 °C until tested for virus. RK-13
cells were used in attempts to recover both herpesvirus hominis and rubella
viruses. Fluids were passed at least twice in these cells and observed over 14-day
periods before being discarded as 'virus-free'.
Stained preparations of rabbit blastocysts
Six and a half days after mating, blastocysts were exposed by gently tearing
open the uterus with forceps. The blastocysts, lying free in the uterus, were
removed using a sterile spoon and fixed immediately in absolute methanol. A
proportion of blastocysts were not fixed but were minced in an M.S.E. blender
and inoculated into cultures of RK-13 cells to detect virus. Fixed blastocysts
were dissected open with mounted needles under low-power microscopy to give
a flat, star-shaped preparation (Moog & Lutwak-Mann, 1958). After air-drying
overnight the blastocysts were stained in Mayers acid haemalum for 25 min and
blued in tap water. Finally, the blastocysts were dehydrated in two changes of
2-ethoxyethanol, cleared in euparol essence and mounted in euparol.
Triethylenemelamine
Compounds
2,4,6-triethyleneimine-l,3,5-triazine (I.C.I. Ltd., Wilmslow) was prepared
freshly for use as a solution containing 1 mg/ml of the compound in deionized
water.
Trypan blue
A 10 % (w/v) solution was prepared in deionized water using a mixture of 5 g
of Gurr's trypan blue (G. T. Gurr Ltd., London, lot number 12409) and 5 g of
B.D.H. trypan blue (British Drug Houses Ltd., London, lot number 685991/
580702).
19-2
288
J. S. OXFORD & R. N. P. SUTTON
RESULTS
Pre-implantation stages of pregnancy
(a) Rabbits
Variations in the shape and size of the embryonic disc of rabbit blastocysts
from control animals inoculated intravenously with RK-13 cells and killed 6^
days after mating are shown in Plate 1. The majority of blastocysts had a relatively large embryonic disc with a regular outline (Plate Ib). A low percentage
(5-10 %) had discs at an earlier development stage with an indefinite outline and
more dispersed cells (Plate 1 a) or, alternatively, were at a later stage with primitive streak rudiments present (Plate 1 c). In this control group of rabbits, four of
the seventy-five blastocysts examined were abnormal, with small disorganized
embryonic discs (table 1).
Table 1. The effect on pre-implantation blastocyst of inoculation of rabbits
in early pregnancy
Material inoculated
'Porterfield' rubella*
(105 TClD50/ml)
Control (RK-13 cells) |
Triethylenemelamine
No. of
rabbits
inoculated
No. of
corpora
lutea
Total no.
No. of
of
abnormal
blastocysts blastocysts
%of
abnormal
blastocysts
104
96
8-3%
80
9
75
9
5-3%
1000%
* 5 ml. volumes inoculated intravenously on pregnancy days 1-5.
f 0-5 mg/kg inoculated intravenously on day 3 of pregnancy.
In contrast, all nine blastocysts removed from a rabbit treated with 0-5 mg/kg
of triethylenemelamine, were abnormal (Plate Id). These blastocysts had very
small, disorganized embryonic discs with degeneration granules.
In the group of rabbits inoculated intravenously with 105 TClD50/ml of the
Porterfield strain of rubella virus (Table 1), eight abnormal blastocysts were
noted out of a total of ninety-six examined. Four of these abnormal blastocysts
were from a single animal and the remaining four blastocysts from three
different rabbits. In each case the embryonic disc was present on the blastocysts but poorly developed and disorganized, as noted in the four abnormal
blastocysts from the control group of animals. As in the control group, most of
the blastocysts examined from the rabbits inoculated with rubella virus had
relatively large embryonic discs with a regular outline (Plate \b). One blastocyst from each rabbit was used for rubella virus recovery experiments; no virus
was recovered from any of these blastocysts.
(b) Hamsters
Eleven hamsters were inoculated intranasally with rubella virus on the second
day of pregnancy and the foetuses examined for abnormalities on day 14 of
J. Embryo I. exp. Morph., Vol. 20, Part 3
PLATE 1
Fig. A. Early stage of development, Embryonic disc with rather indefinite outline. Cells
of disc dispersed.
Fig. B. Intermediate stage of development. Circular disc with a regular, smooth outline.
Fig. C. Later stage of development. Primitive streak rudiments present in posterior part of
disc.
Fig. D. Degenerating embryonic disc of blastocyst. Rabbit inoculated with triethylenemelamine (0-5 mg/kg).
Magnification 60 x
J. S. OXFORD & R. N. P. SUTTON
facing p. 288
Rubella teratogenesis
289
pregnancy (Table 2). No grossly abnormal foetuses were noted out of a total of
eighty foetuses examined. In addition, rubella virus was not recovered from the
eleven foetuses tested, although rubella virus (> 102 TClD50/ml) was recovered
from the lungs of all the inoculated mothers.
Post-implantation stages of pregnancy
(a) Rabbits
Table 3 shows the effects of inoculating rabbits at varying stages of pregnancy
with control tissue culture fluids, viruses or embryopathic agents such as trypan
blue. In control rabbits inoculated with virus free RK-13 cells and killed on
pregnancy day 18, 4-9 % of foetuses were in process of resorption; no grossly
abnormal foetuses were noted in the total of fifty-eight examined. Similar results
were obtained in the group of thirteen rabbits inoculated with 105 TClD50/ml of
the Judith strain of rubella virus. No grossly abnormal foetuses were detected in
a total ninety-eight examined and the resorption rate was 4-9 %. In addition,
rubella virus was not recovered from the forty foetuses tested.
Table 2. Effect on hamster foetal development of inoculation of mother
with rubella virus
Material
inoculated
No. of Days after mating
pregwhen mother:
nant
A
t
\ Total no.
of live
ham- Inoculated Killed foetuses
sters
No. of
live
No. of
but ab- resorpnormal
tion Resorption
foetuses sites
(%)
2
14
11
80
Judith rubella*
0
(105TClD50/ml)
19
118
0
8
14
Judith5 rubella*
(10 TClD50/ral)
10
66
0
2,8
14
Control
(104 RK-13 cells/ml.)
* 0-5 ml. volumes inoculated intranasally.
2
2-4%
13
9-9%
4
5-7%
In contrast, the resorption rate in rabbits inoculated intramuscularly with a
total of 900 mg/kg of trypan blue was 50 %. A high rate of resorption (27-3 %)
was noted in rabbits inoculated intravenously with 106 TClD50/ml of herpesvirus
hominis. Herpesvirus (> 1020 TClD50/ml) was recovered from the living foetuses
and the corresponding placentas of two of the four rabbits inoculated. These
live foetuses were examined for congenital defects but were grossly normal.
(b) Hamsters
Nineteen hamsters were inoculated intranasally on the 8th day of pregnancy
with 105 TClD50/ml of the Judith strain of rubella and killed 14 days after mating
(Table 2). The number of resorptions was thirteen and the total number of live,
r
11
18
7, 9, 10
30
18
3, 7, 10, 14
58
16
33
25
10, 14
63
98
Total no.
of live
foetuses
121
Killed
•\
No. of
corpora
lutea
3, 5, 7, 10, 14 18
Inoculated
A
Days after mating
when mother:
0
0
0
0
No. of
live but
abnormal
foetuses
* 5 ml volumes inoculated intravenously and 1 mlvolumes intranasally.
f Inoculated intramuscularly.
13
Judith rubella
(105TClD50/ml)*
4
Herpesvirus hominis
(106TClD50/ml)*
Control-RK-13 cells
6
(10* cells/ml)*
3
Control (trypan
blue 300 mg/kg per dose)f
Material
inoculated
No. of
pregnant
rabbits
11
3
6
5
No. of
resorption
sites
50%
4-9%
27-3%
4-9%
(%)
Resorption
Table 3. Effect on rabbit foetal development of inoculation of mother with viruses or trypan blue
H
cH
GO
z
a
o
*>
*~\
•n
X
o
O
Rubella teratogenesis
291
grossly normal foetuses 118. Forty of these foetuses were minced and the resulting fluids tested for rubella virus; no virus was recovered. However, rubella
virus (> 1020 TClD50/ml) was recovered from the lungs of all nineteen mothers.
Ten control hamsters were inoculated with virus-free RK-13 cells and the total
number of live foetuses on pregnancy day 14 was sixty-six, with four resorption
sites.
DISCUSSION
Viruses and chemicals which are teratogenic to the mammalian foetus commonly exert their activity during the stage of organogenesis when the various
organs of the body are differentiated. The interruption of foetal development by
a virus may be caused directly by an actual infection of the embryo, or indirectly
by a toxaemia, pyrexia or a placental change produced by the virus in the mother.
In the present study the pre-implantation rabbit blastocyst was used as a model
with which to investigate any such indirect teratogenic action of rubella virus.
The rabbit blastocyst at this stage is very sensitive to the toxic action of a number of agents (Lutwak-Mann & Hay, 1962). Thus in the present study all blastocysts removed from a pregnant rabbit inoculated with triethylenemelamine
showed degenerative changes (Adams et ah 1961). In contrast, in the group of
rabbits inoculated with rubella virus, only 8-3 % of the blastocysts were
abnormal. Hafez & Rajakoski (1964) examined blastocysts from domestic
rabbits at 144 h post coitum and estimated that 8 % were degenerating or undersized. In another investigation Adams (1960) studied the loss of ova before
implantation in 126 rabbits and noted that 9-7-11-2 % of ova were lost before
implantation. The rate of abnormality obtained in the present study in the
rabbits inoculated with rubella virus is therefore within the range expected in
control, untreated animals and therefore no cytotoxic product of rubella virus
multiplication in tissue cultures was detected. Rubella virus multiplies in RK-13
cells and produces a soluble, complement-fixing antigen (Schmidt & Lennette,
1965). Preliminary studies with infected tissue-culture fluids containing rubella
complement-fixing antigen (titre 1-8) have also failed to demonstrate any product with a cytotoxic action on the rabbit blastocyst (Oxford, unpublished). It
would be of interest to use the blastocyst technique with other viruses implicated
as teratogenic agents, such as influenza (Coffey & Jessop, 1959; Saxen et ah 1960),
vaccinia (Wielenga, Van Tongeren, Ferguson & Van Rijssel, 1961) and mumps
(Ylinen & Jarvinen, 1953), which produce high concentrations of relatively low
molecular weight complement-fixing antigens.
No embryopathic effect of rubella virus when inoculated at the post-implantation stages of pregnancy in rabbits or hamsters was detected in the present study.
In contrast both trypan blue and herpesvirus hominis were shown to have an
embryocidal effect in rabbits, and herpesvirus was recovered from foetal material
following intravenous inoculation of the mother. A viraemia follows inoculation
of this virus in rabbits and Beigeleissen & Scott (1958) have reported trans-
292
J. S. OXFORD & R.N. P. SUTTON
placental infection of rabbit foetuses. Similarly, herpesvirus hominis has been
shown to cause transplacental infection of the human foetus resulting in foetal
death or lesions (Beigeleissen, Scott & Joel, 1962). The negative finding with
rubella in the present study may reflect the apparent absence of a prolonged
viraemia in the hamster and rabbit following infection with the virus. Rubella
virus produces subclinical infection in the rabbit and hamster following intranasal
inoculation, and virus is most frequently recovered from the lungs of these
animals but only rarely from the blood (Oxford, 1967). In contrast, rubella virus
produces a viraemia in rhesus monkeys following intranasal or subcutaneous
injection and virus can be recovered from the conceptus (Parkman et al.
1965). More recently Cotlier et al. (1968) have described a raised incidence
of neonatal deaths in the offspring of rats which had been injected with rubella
virus during pregnancy; no details were given of any virus multiplication in the
mother rats.
Previous studies have suggested that the production of a viraemia may be an
important prerequisite to transplacental passage of viruses. Thus Uhr and his
colleagues (1963) investigated the passage of a small bacteriophage ( 0 X 174)
across the placenta of the guinea-pig, and noted that transfer of virus only
occurred when the concentration of virus in the mother's blood was greater than
107 particles/ml. Experiments would therefore appear to be indicated using
stocks of rubella virus which have been concentrated to produce higher infectivity titres. In addition, rubella virus grown on cells from other species such as
chick fibroblast cells, grivet-monkey kidney cells or human embryo lung cells
may give different results. An alternative explanation for the apparent absence
of effect of rubella virus on foetal development in the rabbit and hamster may
be that the embryonic cells have no virus receptors for the virus or produce interferon which suppresses rubella virus growth. Preliminary studies have suggested
that even when rubella is inoculated directly into the amniotic area in utero, no
virus multiplication takes place (Oxford, unpublished). In contrast, hamster
and rabbit foetal lungs removed at this stage of pregnancy and grown as organ
cultures support the growth of rubella virus in vitro (Oxford & Schild, 1967).
SUMMARY
1. Intravenous injection of rubella virus had no detectable effect on the preimplantation rabbit blastocyst. A cytotoxic agent, triethylenemelamine, caused
marked degeneration of blastocysts.
2. Rubella virus inoculated intranasally or intravenously in rabbits and
hamsters at post-implantation stages had no effect on foetal development.
Herpesvirus hominis or trypan blue caused an increased rate of foetal resorption
in rabbits.
3. The use of the blastocyst technique to investigate any teratogenic action
of other viruses is discussed.
Rubella teratogenesis
293
RESUME
Action du virus humain de Vherpes et du virus de la rubeole sur les stades
gestatifs de pre- et de post-implantation chez des animaux de laboratoire
1. L'injection intraveineuse de virus de la rubeole n'a pas eu d'effet decelable
sur le blastocyste non implante de lapin. Un agent cytotoxique, la triethylenemelamine, a provoque une degenerescence marquee des blastocystes.
2. Le virus de la rubeole inocule par voie intranasale ou intraveineuse a des
lapins et des hamsters n'a pas eu d'action sur le developpement foetal. Le virus
humain de l'herpes ou le bleu trypan ont provoque un accroissement du taux de
resorption foetal chez le lapin.
3. On discute l'utilisation de la 'technique des blastocystes' pour etudier
l'action teratogene d'autres virus.
We would like to thank Professor C. H. Stuart-Harris for help and criticism during the
study and Drs C. Lutwak-Mann and M. Hay for demonstrating the blastocyst technique.
The careful help of Mr W. Low, Senior Technician, Sheffield University Animal Laboratories,
and Lynda Bebbington and Jennifer Waine is gratefully acknowledged. The study was aided
by a grant from the National Fund for Research into Poliomyelitis and other Crippling
Diseases.
REFERENCES
C. E. (1960). Prenatal mortality in the rabbit Oryctolagus cuniculus. J. Reprod.
Fertil. 1, 36.
ADAMS, C. E., HAY, M. F. & LUTWAK-MANN, C. (1961). The action of various agents upon
the rabbit embryo. /. Embryol. exp. Morph. 9, 468.
ADAMS,
ALFORD, C. A., NEVA, F. A. & WELLER, T. H. (1964). Virologic and serologic studies on
human products of conception after maternal rubella. New Engl. J. Med. 271, 1275.
BEIGELEISSEN, J. Z., & SCOTT, L. V. (1958). Transplacental infection of foetuses of rabbits
with Herpes Simplex Virus. Proc. Soc. exp. Biol. Med. 97, 411.
BEIGELEISSEN, J. Z., SCOTT, L. V. & JOEL, W. (1962). Further evidence of fatal infection with
Herpes Simplex virus. Am. J. clin. Path. 37, 289.
COFFEY, V. P. & JESSOP, W. J. E. (1959). Maternal influenza and congenital deformities—a
prospective study. Lancet ii, 935.
COTLIER, E., Fox, J., BofflGiAN, G., BEATY, C. & Du PREE, A. (1968). Pathogenic effects of
rubella virus on embryos and newborn rats. Nature, Lond. 217, 38.
FERM, V. H. & KILHAM, L. (1965). Histopathologic basis of the teratogenic effects of H-l virus
on hamster embryos. J. Embryol. exp. Morph. 13, 151.
GIBSON, J. P., STAPLES, R. E. & NEWBERNE, J. W., (1966). Use of the rabbit in teratogenicity
studies. Toxic, appl. Pharmac. 9, 398.
HAFEZ, E. S. E. & RAJAKOSKI, E. (1964). Growth and survival of blastocysts in the domestic
rabbit. I. Effect of maternal factors. / . Reprod. Fertil. 7, 229.
INGALLS, T. H., PLOTKIN, S. A., MEYER, H. M. & PARKMAN, P. D. (1967). Rubella: epidemiology, virology and immunology. Am. J. Med. Sci. 253, 349.
KAY, H. E. M., PEPPERCORN, M. E., PORTERFIELD, J. S., MCCARTHY, K. & TAYLOR-ROBINSON,
C. H. (1964). Congenital rubella infection of a human embryo. Br. med. J. ii, 166.
C. & HAY, M. F. (1962). Effect on the early embryo of agents administered
to the mother. Br. med. J. ii, 944.
MOOG, F. & LUTWAK-MANN, C. (1958). Observations on rabbit blastocysts prepared as flat
mounts. J. embryol. exp. Morph. 6, 57.
LUTWAK-MANN,
294
OXFORD,
J. S. OXFORD & R. N. P. SUTTON
J. S. (1967). Growth of rubella virus in small laboratory animals. /. Immunol. 98,
697.
J. S. & SCHILD, G. C. (1966). Growth of rubella virus in the golden hamster (Mesocricetus auratus). Virology 28, 780.
OXFORD, J. S. & SCHILD, G. C. (1967). The evaluation of antiviral drugs for rubella virus
using organ cultures. Arch. ges. Virusforsch. 22, 349.
PARKMAN, P. D., PHILLIPS, P. E. & MEYER, H. M. (1965). Experimental rubella virus infection
in pregnant monkeys. Am. J. Dis. Child. 110, 390.
SAXEN, L., HJELT, L.. SJOSTEDT, J. E., HAKOSALO, J., & HAKOSALO, H. (1960). Asian influenza
during pregnancy and congenital malformations. Ada. path, microbiol. scand. 49, 114.
SCHMIDT, N. J. & LENNETTE, E. H. (1965). The complement fixing antigen of rubella virus.
Proc. Soc. exp. Biol. Med. 121, 243.
UHR, J. W., DANCIS, J., FINKELSTEIN, M. S. (1963). Passage of bacteriophage 0X-174 across
the placenta in guinea pigs. Proc. Soc. exp. Biol. Med. 113, 391.
WIELENGA, G., VAN TONGEREN, H. A. E., FERGUSON, A. H. and VAN RIJSSEL, Th. G. (1961).
Prenatal infection with vaccinia virus. Lancet i, 258.
YLINEN, O. & JARVINEN, P. A. (1953). Parotitis during pregnancy. Acta. obstet. gynec. scand.
32, 121.
ZIMMERMANN, W., GoTTSCHEWSKl, G. H. M., FLAMM, H. & KUNZ, C. H. (1963). Experimentelle Untersuchungen iiber die Aufnahme von Eiweiss Viren und Bakterien wahrend der
Embryogenese des Kaninchens. Devi Biol. 6, 233.
OXFORD,
(Manuscript received 20 February 1968)