Download Vertical Transmission of Murine Cytomegalovirus

J. gen. ViroL 0979), 42, 621-625
Printed in Great Britain
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Vertical Transmission of Murine Cytomegalovirus
(Accepted 3 October 1978)
SUMMARY
Congenital abnormalities induced by murine cytomegalovirus have previously
been suggested to be an indirect effect of maternal illness as no infectious virus has
been isolated from the foetus. However, in this article we report that latent virus
detectable by immunofluorescence and in situ hybridization, may frequently be
present in foetal tissues.
Congenital infections with cytomegalovirus are believed to be one of the major causes of
human birth defects including mental retardation, microcephaly, epilepsy, blindness and
cerebral palsy (Stern et al. 1969; Hanshaw, I97O). As many as I ~ of all infants may be
born with CMV infection (Stern & Tucker, 1973) and although many of these infections are
asymptomatic, they too may represent significant but subtle effects on the CNS preventing
full development of a child's potential (Stern et aL I969; Hanshaw, 1971).
Several investigators have examined the mouse cytomegalovirus as a model for the
human infection. The cytomegaloviruses as a group are fairly host-specific viruses, characterized by their ability to initiate long-term persistent infections which are often accompanied
by the shedding of virus in tissue secretions for considerable lengths of time. The mouse
virus, like its human counterpart, is known to cause foetal injury which is manifested as
decreased litter size, abortion or the delivery of stillborn macerated foetuses (Mannini &
Medearis, 1961; Medearis, 1964) after inoculation of mice at the time of implantation
(Neighbour, I976) or during pregnancy (Mannini & Medearis, I96I ; Johnson, 1969).
However, a major difference has been suggested in the course of congenital infections by
the human and murine viruses. In the human case, the virus has frequently been isolated
from the foetus and is believed to be transmitted across the placenta from the maternal blood
system (Potter, 1957; Wcller, 1970. In addition it has been suggested that reactivation of
latent virus in the cervix or urinary tract may be important (Montgomery et al. 1970, or
that venereal transmission in semen may occur (Lang & Kummer, 1972). However, in
persistent murine infections no virus has been detected in the foetus. Medearis (1964) and
Johnson (1969) were unable to demonstrate intra-uterine infection with MCMV either during
acute infection of the mother, i.e. when a generalized viraemia was occurring, or during
persistent infection when the virus is probably localized in salivary glands, kidneys and
possibly the lymphocytes (Olding et al. 1975, 1976). They therefore suggested that the
abnormalities were an indirect result on the foetus of maternal illness or a change in the
placental environment. These results indicated a prime limitation in the use of the murine
system as a model for the human infection. However, in this article we report that we have
been able to detect the presence of latent MCMV in embryos from persistently-infected mice
by immunofluorescence and in sltu hybridization.
Several 2 to 3 week old female mice (SWR, Jackson Laboratories, Bar Harbor, Maine)
were injected intraperitonealty with lO4 p.f.u, of tissue-culture passaged MCMV (Smith
strain; Hudson et al. 1976) and were observed periodically for signs of illness. No obvious
symptoms were detected but high titres of virus were found in the salivary glands of selected
mice up to 9° days after infection. Over the next few months, the offspring of these infected
mice were occasionally observed to be runty with ruffled coats which had an oily appearance.
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In addition, litter sizes were repeatedly small and the new-borns had diarrhoea and running
eyes. These effects were observed sporadically. Some litters appeared to be normal although
some of the offspring had ruffled coats and thus may not have been completely healthy.
Stocks of uninfected animals did not exhibit these effects, except for small litter sizes which
were observed occasionally.
Approximately one year after intraperitoneal inoculation, three pregnant mice were
sacrificed and found to contain abnormal uteri. Two of these contained only two embryos
as opposed to the normal complement of 7 to 14 while the third contained a single large
dead foetus. Each pair of viable embryos was treated by standard procedures for preparing
primary embryo cultures (Younger, 1954), and passaged for several generations. Extreme
care was taken while removing the embryos from the mothers and in their subsequent
cultivation to ensure that contamination with MCMV from either the laboratory or the
maternal tissues did not occur. The two lines were kept separate, and designated EM-2
and EM-3. The single large foetus (EM-1) was discarded as it did not yield viable cells and
had no infectious virus associated with it.
At passage 4 the EM-3 cells developed a characteristic MCMV-induced cytopathic effect
(c.p.e.). On examination of the medium of these cultures under the electron microscope,
herpes-like virions were detected with the multicapsid structure unique to MCMV (Hudson
et al. I976), indicating that activation of latent MCMV had occurred. In contrast, there was
no infectious virus associated with EM-2 cells from passage 1 to passage 7, after which the
line degenerated without any obvious production of c.p.e. Virus-like particles were not
detected in the medium, nor in cell lysates of EM-z cells and therefore the search was
extended to include the possibility that latent MCMV was present in these cells. Indirect
immunofluorescence and in situ hybridization tests were performed on EM-2 cells fixed on
coverslips from early passages, while solution hybridization was conducted using cells from
later passages when more material could be obtained.
The result of an indirect immunofluorescence test is shown in Fig. I. The antisera for
these experiments were prepared in rabbits against total infected cell lysates as has been
described elsewhere (Chantler & Hudson, 1978). The sera were adsorbed thoroughly against
mock-infected cells before use and gave minimal background fluorescence in uninfected cells
(Fig. I a) but striking fluorescence with tertiary mouse embryo cells productively infected
in vitro (Fig. I b). The result for EM-2 cells is shown in Fig. I (c) and (d). These cells have a
characteristic 'stringy' appearance which readily distinguishes them from normal EM cells
during early passage. Although not all the EM-2 cells exhibited fluorescence, a significant
number showed distinct membrane fluorescence as well as a small amount of granular
fluorescence within the cell. In addition to this, occasional foci of bright fluorescence were
observed. These results indicated that MCMV was indeed present in these ceils and that
a low level of virus expression was occurring in discrete areas.
The search for the virus genome was conducted initially by D N A - D N A hybridization in
solution, using D N A extracted from EM-2 cells at passage 7, and 12sI-labelled MCMV D N A
(Misra et al. 1978). However, because only a small amount of EM-2 D N A was obtained,
only 50 #g of unlabelled DNA could be used per assay and no virus sequences were detected.
This may have been due to the lack of sensitivity of the experiment due to the low concentration of cellular DNA, or alternatively, to a selective reduction of cells carrying the virus
genome during successive passage of the EM-2 line. To surmount these problems, in situ
hybridization was performed on coverslips of EM-2 cells obtained from passages 2 and 3The protocol for this technique is described elsewhere (Haase et al. 1977) and outlined in
the legend to Fig. 2. Hybridization was carried out with 12~I-labelled MCMV probe D N A
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Fig. ~. An indirect immunofluorescence test using rabbit antiserum to MCMV proteins and
FITC-conjugated goat anti-rabbit IgG, examined on a Leitz Wetzlar fluorescence microscope.
Original magnification x 4o. (a) Mock-infected; (b) lyric infection (zo h); (c) and (d)EM-z cells.
(by,
Fig. 2. In situ hybridization of 12H-labelled MCMV D N A to mouse embryo tissues: (a) mockinfected; (b) 2o h after infection in vitro; (c) and (d)EM-2 cells. Hybridization was performed on monolayers of cells fixed in o'2 N-HC1 on coverslips. After pre-treatment with ribonuclease, the D N A was
denatured in 95 ~ formamide o'I x SSC at 65 °C for 2 h. Hybridization was in 5o ~ formamide
4 x SSC at 45 °C for 72 h using 5/zl of a~SI-labelled MCMV D N A (0-225/zg/ml: ~.6 x I o 7 ct/min/l~g).
The coverslips were then washed in acetate buffer (3o mM-sodium acetate, pH 4'5, Ioo mM-NaCI,
2 mM-ZnC12) and treated with IO units/ml of $1 nuclease at 5o °C. They were coated with Ilford
K2 emulsion and after being exposed for I week, were developed and stained with Giemsa.
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at 45 °C in 5o % formamide and autoradiography performed on the coverslips for I week.
Embryo cells from uninfected mice, and cells infected with MCMV in vitro and harvested
at 2o h p.i. were used as controls (Fig. 2a and b). The results show very low numbers of
background grains in uninfected cells (a) while in contrast the infected cells at 2o h.p.i.
contained many grains concentrated in the nucleus although also present in the surrounding
cytoplasm (b). In comparison, the results for EM-2 cells are shown in Fig. 2(c) and (d).
In (e), a single cell with cytoplasm barely visible can be seen to contain a high number of
grains confined within the nuclear membrane. In (d) another EM-2 cell is shown with 4 or 5
grains in the nucleus and essentially none in the surrounding area. No foci of active replication could be identified by in situ hybridization as, during the procedures involved, a large
number of the cells detach and only isolated cells remained on the coverslip. However,
approximately the same proportion of cells as exhibited granular fluorescence were found to
contain intra-nuclear grains by in situ hybridization.
These preliminary results in contrast to those of other workers (Medearis, I964; Johnson,
I969) show that vertical transmission of MCMV from mother to foetus does occur. However, the virus appears to exist in the foetus in a latent form and has thus previously escaped
detection. In the EM-3 cell line, the virus was reactivated by successive passage of the cells
in vitro, but this may be an unusual occurrence. In the case of the EM-2 cells, infectious virus
was not detected at any stage although significant virus gene expression was found by
immunofluorescence and in situ hybridization. These results indicate that the asserted
limitations of the murine system as a model for human cytomegalovirus infection may be
unjustified.
Jordan et aI. 1977 recently reported the reactivation of MCMV from latently infected mice
using immunosuppression with anti-lymphocyte serum and corticosteroid. In this study,
97 % of mice inoculated subcutaneously with MCMV could be shown to harbour a latent
infection some 4 to 8 months later. This finding, taken in conjunction with the possibility
of impaired maternal responsiveness to foreign antigen during pregnancy (Hellstrom et al.
1969), indicates a possible mechanism for reactivation of the virus under natural conditions.
In addition, the genome of MCMV has been detected recently in germ-line cells of testicular
origin by in situ hybridization (F. J. Dutko & M. B. A. Oldstone, personal communication).
This suggests two possible mechanisms for transmission of the virus to the foetus; transplacental passage of a reactivated virus, or direct vertical transmission through a germ-cell.
We cannot distinguish between these possibilities at present.
These findings, together with our detection of MCMV in viable embryos from latently
infected mothers, have considerable implications for the proposal to vaccinate humans with
live attenuated virus in an attempt to prevent CMV-induced birth defects. Indeed in preliminary results from a current experiment, we have found that both virulent strains of MCMV
(passaged in mouse sub-maxillary gland) and attenuated strains (passaged in cell-culture)
have very similar effects on pregnancy wastage in the offspring.
We thank Dr A. T. Haase for supplying details of his protocol for in situ hybridization.
This work was supported by grant MA-4762 from the Medical Research Council of Canada.
Division o f Medical Microbiology
University o f British Columbia
Vancouver, B.C. V6T I W5
Canada
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J. K. CHANTLER
VIKRAM MISRA
J. B. HUDSON
Short communications
625
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