Download Infection of Cultured Early Mouse Embryos with Semliki Forest and

J. gen. Virol. (1986), 67, 1091-1098.
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1091
Key words: SFV/ R V/mouse embryos
Infection of Cultured Early Mouse Embryos with Semliki Forest and
Rubella Viruses
By A N N E T T E
M. H E A R N E , M. A I D E E N O ' S U L L I V A N AND
G R E G O R Y J. A T K I N S *
Department of Microbiology, Moyne Institute, Trinity College, Dublin 2, Ireland
(Accepted 20 February 1986)
SUMMARY
Early mouse embryos at the four- to eight-cell stage or the blastocyst stage could be
infected with the A7 strain of Semliki Forest virus (SFV) after the removal of the zona
pellucida, either by Pronase treatment or following hatching of blastocysts. With SFV,
rapid virus production and eventual cytolysis resulted from infection at either stage.
For four- to eight-cell embryos the cytopathic effect was delayed and a proportion of
embryos developed to the blastocyst stage. Four- and eight-cell embryos could not be
infected with rubella virus (RV), even after removal of the zona pellucida. RV infection
of zona-free blastocysts resulted in a productive but non-cytolytic infection which did
not affect embryonic development to the early egg-cylinder stage. RV did not multiply
in inner cell mass cells isolated from embryos at the blastocyst stage, although SFV did
multiply in such cells.
INTRODUCTION
Many togaviruses are known to infect the developing foetus and produce abortions or
teratogenic defects in the offspring. Such viruses include Semliki Forest virus (SFV; Atkins et
al., 1982; Milner & Marshall, 1984; Milner et al., 1984), rubella virus (RV; Gregg, 1941 ; Siegel
& Greenberg, 1960), bovine viral diarrhoea virus (Done et al., 1980), border disease virus
(Gardiner & Barlow, 1972) and Japanese encephalitis virus (Chaturvedi et al., 1980). In general,
damage to the foetus is greatest when the viral infection occurs in the early stages of pregnancy
during gastrulation and organogenesis (Fuccillo & Sever, 1973; Sugamoto & Miura, 1982).
The A7 strain of SFV has previously been shown to be lethal for the developing mouse embryo
when the mother is inoculated on day 8 of pregnancy, although the mother survives the infection
(Atkins et al., 1982; Milner & Marshall, 1984). The route of infection appears to be invasion of
the placenta by virus present in the maternal blood followed by invasion of the foetus 1 or 2 days
later (Milner & Marshall, 1984). However, little is known about the interaction between embryo
and virus, a major obstacle being the inaccessibility and small size of the embryo at the early
developmental stages. We have previously shown that outgrowths of ectoplacental cone
trophoblast cells from 8-day embryos are susceptible to infection with the A7 strain of SFV, but
are less susceptible to infection with the neurovirulence mutant M103. This mutant does not
infect the developing embryo following infection of the mother but does confer post-natal
immunity to the offspring (Atkins et al., 1982).
In the present study, we have continued this work by developing a system for infecting mouse
embryos in vitro. Pre-implantation mouse embryos can be grown in vitro from the two-cell stage
(36 to 48 h post-conception; p.c.) through the morula stage at 80 to 100 h p.c. and the blastocyst
stage (3 to 3.5 days p.c.) in a simple medium (Whittingham, 1971), to the post-implantation early
egg-cylinder stage (5.5 days p.c.; Hsu, 1979). When an embryo reaches the blastocyst stage it
hatches from the zona pellucida, an acellular glycoprotein coat surrounding it, and establishes
direct contact with the mother through the process of implantation. Here we have initially used
the A7 strain of SFV as a model for embryonic infection and then extended the system to RV.
Embryos at early and late pre-implantation stages were infected and monitored for virus
production, effects on embryonic development in vitro and role of the zona pellucida.
0000-6974 © 1986 SGM
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A . M . HEARNE, M. A. O'SULLIVAN AND G. J. ATKINS
METHODS
General, The methods for growing cloned stocks of SFV and the plaque assay on BHK cells have been previously
described (Atkins et al., 1974). Stocks of the A7 strain of SFV were tested for virulence in pregnant mice on day 8
of pregnancy and their virulence was as previously described (Atkins et al., 1982).
The Therien strain of RV and the host Vero cells were a gift from Dr R. Pettersson, Department of Virology,
University of Helsinki, Finland. Virus was harvested from Vero cells grown in microcarrier culture (Cytodex,
Pharmacia) in BHK-21 medium (Gibco) and plaque-assayed on Vero cells in 60 mm plastic dishes. Plaques were
visible 5 to 6 days later, after staining monolayers overnight with 0-1% neutral red solution in phosphate-buffered
saline (PBS).
Collection andculture of embryos. A line of Q/Fa mice, selected for high body weight (Hayes & McCarthy, 1976),
was used for embryo collection. Prepubertal females (21 to 25 days) were induced to superovulate by an
intraperitoneal (i.p.) injection of 5 IU pregnant mare's serum gonadotrophin (Folligon; Intervet, Cambridge,
U.K.) followed 46 h later by an i.p. injection of 2.5 IU of human chorionic gonadotrophin (hCG; Chorulon;
Intervet). After the hCG injection each female was placed in a cage with a BALB/c male and examined on the
following morning for a copulation plug (day 1 of pregnancy). Early on day 3 of pregnancy females were killed by
cervical fracture and the oviducts removed. Four- to eight-cell embryos were flushed from the oviducts with PBS
and pooled in medium 16 (Whittingham, 1971). Morphologically normal embryos were transferred through at
least four changes of medium 16 before transfer to 50 ~1 droplets of medium 16 for culture under oil (light liquid
paraffin, wt./ml 0.830 to 0.860 g, BDH) at 37 °C in a humified CO2 incubator (Whittingham, 1971). Four- to eightcell embryos reached the blastocyst stage after 40 to 50 h. Embryos at this stage were transferred to a serumcontaining medium (Hsu, 1979) for outgrowth and development to the early egg-cylinder stage (after 4 days).
Removal of the zona pellucida with Pronase. A 1~ stock of Pronase (Streptomyees griseus protease, Sigma) was
incubated at 37 °C for 1 h to allow autodigestion, before storage in 50 ~tl aliquots at - 20 °C. Embryos were washed
in PBS before treatment with Pronase for 2 to 3 rain at room temperature, as described by Mintz (1962). In general,
early embryonic stages required longer enzyme treatment than blastocyst stage embryos. Zona-free embryos were
incubated for 1 to 2 h in medium 16 to allow membranes to recover before further use.
Isolation of inner cell mass (1CM) cells. The immunosurgery technique described by Nichols & Gardner (1984)
was used. Anti-mouse antibody was raised in rabbits to day 14 foetal mouse tissue, and rat serum was used as a
source of complement. Rabbit and rat sera were stored in 50 ~tl aliquots at - 20 °C. Zona-free blastocysts were first
incubated in diluted antiserum for 30 rain at 37 °C and then, after three washes in PBS, for a further 30 rain in
diluted rat serum at 37 °C for trophoblast lysis. Following a rinse in medium 16, blastocysts were transferred to
fresh droplets of medium 16, and the ICM cells were dissected free of the trophoectodermal debris using a glass
micropipette. Isolated ICMs were incubated in Hsu's medium overnight before infection with virus.
Virus injection of embryos and isolated 1CMs. Groups of 15 to 20 embryos or ICMs were infected in 50 ~tl culture
medium containing virus at the appropriate dilution. Unless otherwise specified, the multiplicity of infection was
10~ p.f.u, per embryo or per ICM. After virus adsorption for 1 h embryos or ICMs were washed by passing them
through five droplets of fresh medium, then allocated, as subgroups of five to ten each, to 50 ~tl droplets of culture
medium. The medium was replaced after 5 to 7 h. This and the medium from the final wash were stored at - 70 °C
after dilution in 2 ml BHK medium, to be assayed subsequently for the presence of non-specifically adsorbed
virus; these samples were always negative. At specified times after infection the culture medium was replaced with
fresh medium; the removed medium was stored in 2 ml BHK medium at - 70 °C, before assay for the presence of
virus. Mock-infected control embryos or ICMs were subjected to the same manipulations and medium changes
and their morphological development was compared to that of virus-infected embryos or ICMs.
RESULTS
Injection o f Jour- to eight-cell embryos with S F V
Initial e x p e r i m e n t s i n v o l v i n g infection of e m b r y o s at the four- to eight cell stage w i t h S F V
showed that no infectious virus was p r o d u c e d and e m b r y o n i c d e v e l o p m e n t to the blastocyst
stage was c o m p a r a b l e to that o f u n i n f e c t e d controls. R e m o v a l o f the zona pellucida w i t h P r o n a s e
was therefore carried out. Loss of the zona had no effect on e m b r y o n i c d e v e l o p m e n t to the
blastocyst stage. H o w e v e r , zona-free e m b r y o s t e n d e d to f o r m either m i n i a t u r e blastocysts due to
the loss o f some cells f r o m the c l e a v i n g e m b r y o or gigantic blastocysts due to the fusion o f two or
m o r e embryos. O n transfer to H s u ' s m e d i u m these blastocysts a t t a c h e d and o u t g r e w in the
n o r m a l way. T y p i c a l cultures of z o n a - c o n t a i n i n g and zona-free e m b r y o s are s h o w n in Fig. 1.
E m b r y o s were inoculated with 104 p.f.u. S F V , not an excessive dose since m a t e r n a l v i r a e m i a
reaches 8 x 106 p.f.u,/ml ( G a t e s et aL, 1985) and m a t e r n a l blood is in direct c o n t a c t w i t h the
t r o p h o b l a s t cells of the i m p l a n t e d embryo. Only infection o f zona-free e m b r y o s resulted in a
p r o d u c t i v e infection (Fig. 2). Virus p r o d u c t i o n p e a k e d at 26 h post-infection and c o n t i n u e d until
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Fig. 1. Four- to eight-cell stage embryos with and without zonae pellucidae. (a) Zona-free. (b) Zona
intact. (c) Zona-free embryos form miniature (m) and gigantic (g) blastocysts. (d) After 48 h in culture
embryos with intact zonae develop into blastocysts of approximately equal size. Bar markers represent
100 I~m.
3.0
2.5
@
.~ 2.0
-i 1.5
2
_o 1.0
0.5
Oil0
=~'
lO
20
30
40
50
60
Time post-infection (h)
70
80
90
Fig. 2. Virus production by four- to eight-cell stage embryos infected with 10~ p.f.u./embryo SFV. I-q,
Embryos having intact zonae; n , zona-free embryos.
the embryos were necrotic at 90 h. The first signs of c.p.e, were observed at 67 h. In most
experiments two peaks of virus production occurred but the reason for this is not clear at present.
The proportion of infected embryos developing to the blastocyst stage depended on the titre of
input virus (Table 1). The effect of infection with SFV on four- to eight-cell embryos is shown in
Fig. 3.
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A. M. HEARNE, M. A. O'SULLIVAN AND G. J. ATKINS
a)
?
Fig. 3. Four- to eight-cell embryos 90 h after infection with 10a p.f.u./embryo SFV. (a) An embryo
having an intact zona at the time of infection has hatched from its zona and outgrown; T, trophoblast
cells; ICM, inner cell mass. (b) An embryo which was zona-free at the time of infection is now necrotic.
Bar markers represent 100 lain.
4.0
3.5
3.0
O
2.5
2.0
e~
-~ 1.5
1.0
0.5
0
20
30
40
50
Time post-infection (h)
Fig. 4. Virus production by blastocyst stage embryos infected with 104 p.f.u./embryo SFV. [~,
Embryos having intact zonae; II, embryos hatching from their zonae; O, zona-free embryos.
T a b l e 1.
0
10
Effect of SFV infection on development of four- to eight-cell embryos in culture
Input m.o,i,
(p.f.u./embryo)
0
5 × 103
3 x 104
3 x 106
Proportion at the blastocyst stage
at 50 h post-infection
r
~
"~
Zona-free
Zona intact
18/18
ND*
10/18
23/27
1/30
ND
0/30
ND
* ND, N o t d o n e .
Infection of blastoeysts with SFV
T h e role o f t h e z o n a p e l l u c i d a in p r e v e n t i n g virus infection was also i n v e s t i g a t e d for e m b r y o s
at the blastocyst stage. F o r this purpose three e x p e r i m e n t a l groups o f blastocysts were i n f e c t e d :
firstly, those w h i c h had b e g u n to h a t c h f r o m the z o n a pellucida and thus had a c r a c k or o p e n i n g
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Fig. 5. Infection of blastocyst stage embryos with 104 p.f.u./embryo SFV. (a) Hatching embryos (h). (b)
Embryo having had its zona removed by Pronase treatment. At 48 h after infection embryos having
intact zonae at the time of infection developed into normal outgrowths (c) while the hatching and zonafree embryos were necrotic (d, e). Bar markers represent 100 ~tm.
in the zona; secondly, those that had had their zonae removed by Pronase treatment; thirdly
blastocysts having intact zonae. The results are shown in Fig. 4. Infection of either hatching or
zona-free blastocysts with SFV resulted in rapid virus production which peaked at 20 h postinfection; after 48 h all infected embryos were necrotic. An intact zona pellucida at the time of
exposure to SFV prevented virus infection. The appearance of typical infected blastocysts is
shown in Fig. 5.
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A.M. HEARNE, M. A. O'SULLIVAN AND G. J. ATKINS
12
I
I
I
I
!
lO
8
0
~
4
2
20
40
60
80
Time post-infection (h)
100
1~0
Fig. 6. Virus production by blastocyst stage embryos infected with 104 pX.u./embryo RV. 12],Embryos
having intact zonae; II, embryos hatching from their zonae; 0 , zona-free embryos. Note that the
ordinate has an arithmetic scale.
Table 2. Infection of isolated ICM cells with SFV and R V
Time
post-infection*
(h)
0
16
22
43
Vi.rus production (p.f.u./ICM)
•
"
SFV
RV
0
0
15
0
6 x 104
0
3 x 104
0
* ICMs were infected with 104 p.f.u./ICM.
Infection of cultured early mouse embryos with R V
Initial experiments involving i.p. or intravenous injection of 4- or 8-day pregnant mice with
RV showed that no viraemia was produced from 6 h to 6 days after infection, and embryonic
development was not affected. Subsequent experiments were therefore confined to cultured
embryos.
The experiments described above for SFV were repeated with RV. No infectious virus was
produced following exposure of four- to eight-cell embryos, either with or without their zonae
pellucidae, to RV. The proportion of embryos developing to the early egg-cylinder stage was
equivalent to that of uninfected controls. However, a different result was obtained following
infection of embryos at the blastocyst stage. Although embryos with intact zonae produced no
infectious virus, both hatching and zona-free embryos did undergo a low yielding but productive
infection (Fig. 6). Virus infection had no effect on development of embryos to the early eggcylinder stage.
Infection of isolated ICM cells
I C M cells isolated from embryos at the blastocyst stage were infected with RV and with SFV
as a control. No virus production could be detected in these cells following RV infection,
although a productive infection was obtained with SFV. Intact blastocysts infected in the same
experiment with SFV also produced infectious virus (Table 2).
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DISCUSSION
This study has utilized SFV as a model for togavirus infections of mouse embryos at early
developmental stages. The results show that the zona pellucida protects the early embryo from
SFV and RV infection. Prevention of virus entry by the zona pellucida is not unique to SFV
infection; bovine viral diarrhoea and Akabane viruses (Singh et al., 1982), bluetongue virus
(Bower et al., 1982) and Newcastle disease virus (Glass et al., 1974) are also unable to penetrate
the zona. However, other viruses can readily cross the zona pellucida, for example Mengo
encephalitis virus (Gwatkin, 1963, 1967), western equine encephalomyelitis virus (Gwatkin,
1971) and coxsackie B3 virus (Eaglesome et al., 1980). The reason for the selective passage of
some viruses is not clear but may be related to physical and charge characteristics of the
channels present in the zona pellucida (Gwatkin, 1967, 1971; Sellins & Jenkinson, 1975).
Exposure of zona-free embryos to SFV always resulted in a cytolytic infection. Milner &
Marshall (1984) have found that alphavirus infection of pregnant mothers on day 8 results in a
lethal infection of all embryos but infection on day 5 or earlier results in survival of a proportion
of embryos. In the animal, the rate of development of individual embryos in a group is
asynchronous. On average, implantation begins on day 4, but some embryos will not begin to
implant until later (Rugh, 1968). Thus, while the zona pellucida could protect the embryo in the
uterus from a lethal infection with SFV, it is probably the extent of implantation of individual
embryos in the uterine wall at the time of viral infection which regulates the ability of virus to
infect from the maternal blood.
With their zonae removed, both early and late preimplantation stage embryos were
susceptible to SFV, but the situation with RV was different. Susceptibility correlated with the
blastocyst stage, as has also been found for polyomavirus infection of mouse embryos (Biczysko
et al., 1973). Non-cytolytic infections with RV have previously been reported (Rawls & Melnick,
1966; McCarthy, 1969; Boue, 1969). In cultures established from a large number of cells of
infected human infants and foetuses all the cells of the culture actively produced virus. However,
virus did not produce a direct cytopathic effect but slowed the growth rate and ultimately the
doubling time of these cells (Rawls & Melnick, 1966). In the present study virus production was
not accompanied by c.p.e, and virus infection did not interfere with embryonic development.
An embryo at the blastocyst stage is a hollow ball consisting of two types of cells; trophoblastic
ceils which form the outer layer, to which the ICM cells are attached as a discrete disc on the
inside. The ICM cells of the embryo ultimately form the foetus in the animal. In this study we
found that RV-infected isolated ICMs did not release infectious virus during a 48 h culture
period after infection while these cells were capable of supporting SFV replication. This may
indicate that only the trophoblastic cells are susceptible to RV or that virus must replicate in
trophoblast cells before it can infect the ICM. It is difficult to differentiate between these
possibilities in the present short-term culture system. Longer term culture periods are necessary;
one way to accomplish this would be to transfer RV-infected embryos to pseudo-pregnant
females.
In this model, SFV and RV infections of the developing early embryo in vitro gave extreme
effects; SFV was rapidly cytolytic, while RV infection resulted in low virus production without
cytolysis. To learn more about embryopathic mechanisms we are currently testing mutants of A7
SFV with altered virulence in vivo in this system.
We thank Dr H. Killen and Dr J. Carrol for advice on embryo culture, Ms D. Fahy for rubella virus and the
Medical Research Council of Ireland for financial support.
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