Effects of Influenza, Mumps, and Western Equine Encephalitis Viruses on Fetal Rhesus Monkeys (Macaca mulatta) A. F. MORELAND, J. M. GASKIN, R. D. SCHIMPFF, J. C. WOODARD AND G. A. OLSON University of Florida, J. Hillis Miller Health Center, College of Veterinary Medicine and College of Medicine, Gainesville, Florida 32610
ABSTRACT Pregnant Rhesus monkeys were infected via instillation of influenza, mumps and western equine encephalomyelitis viruses respectively into the amniotic sacs a t approximately 90 days gestation to determine if fetal infections would occur. Virus was recovered from fetal tissues after seven days in 100%of the exposed animals. Thus, the viruses are capable of causing fetal infection. Rhesus monkey fetuses were inoculated with influenza, mumps and WEE viruses by the direct intracerebral route a t approximately 90 days gestation to determine possible teratogenicity of the viruses. Influenza virus caused no malformations or measurable fetal effects. Mumps virus resulted in significant fetal mortality, WEE virus resulted in a 100%incidence of encephalitis and hydrocephalus. Thus,mumps and WEE viruses are teratogens in primates and are potential teratogens of man. Numerous viruses are known to seriously and either congenital malformations or subseaffect neonatal development and the effects of quent neoplasms during childhood.” Perhaps prenatal infections with rubella, herpesvirus the strongest evidence supportive of malforsimplex Type I1 and cytomegalovirus are well mations in higher mammalian species to date established. I t has been long suspected, how- resulted from a study by London et al. (‘75) ever, that congenital malformations and fetal which demonstrated occurrence of hydrocephmortality may be attributable to other pre- alus in fetal rhesus monkeys following intracerebral inoculation of influenza A virus. natal virus infections. The observation that neural tube malforma- It is against this background of conflicting retions were induced in chick embryos inocu- ports that we decided to conduct the present lated with influenza virus was first described studies on influenza virus. by Hamburger and Habel (‘47)and has been Siege1 et al. (‘66) reported that human subsequently confirmed by numerous others. maternal mumps virus infection during the Fetal wastage and abnormalities of the ner- first trimester was associated with increased vous system in mice have been reported by fetal mortality, however, infection of fetuses Fuccillo and Sever (‘73) and hydrocephalus was not demonstrated by virus isolation. has resulted from direct intracerebral inocu- Johnson (’68a) reported hydrocephalus in lation of influenza virus into suckling ham- suckling hamsters following intracerebral insters and mice as reported by Johnson and oculation with mumps virus and Ennis et al. Johnson (‘69). Evidence incriminating influ- (’69) confirmed this work. Kilham and Marenza as a human teratogen and as a predis- golis (‘75)reported induction of hydrocephposing factor for neoplasms has been recently alus in hamsters after inoculation of mumps reviewed by MacKenzie and Houghton (’74). virus into the amniotic sac and St. Geme and While many studies have demonstrated a Van Pelt (’74)described infections in fetal significant relationship, perhaps equally as Received April 27, ‘78. Accepted Jan. 20, ’79. many have not, thus leading them to the conSupported by Contract NS-5-2318from the National Institutes clusion that “probably no direct association of Health. U. S. Public Health Service, Department of Health. exists between maternal influenza infections Education and Welfare. 1
TERATOLOGY(1979) 20: 53-64.
53
54
MORELAND, GASKIN, SCHIMPFF. WOODARD AND OLSON
rhesus monkeys following intravenous inoculation of the mother thus indicating transplacental passage of the virus, London et al. (’71) reported a high rate of fetal mortality in rhesus monkey fetuses inoculated intracerebrally. No malformations occurred in either of the latter two studies, although both reports indicated growth retardation in fetal animals and St. Geme and Van Pelt further observed this in neonates. Thus, there appears to be a clear association between mumps virus and a high rate of fetal mortality. The relationship to congenital malformations is less certain although strongly suggestive. For this reason mumps virus was included in the present study. Probable transplacental infections of human fetuses with western equine encephalitis (WEE) virus have been reported by Medovy (’431, Shinefield and Townsend (’531,Cohen et al. (’53), and Copps and Giddings (’59), however, all were late in gestation (10-12 days prior to parturition) and malformations were not observed. Nevertheless, the potential for infection of mothers early in gestation is great and since WEE virus infections in adult humans are often mild, transient, brief infections which often go unrecognized the probability of this togavirus contributing to the incidence of malformations in humans is of considerable interest and concern. Spertzel et al. (’72) showed transplacental passage of Venezuelan equine encephalitis virus in mice which resulted in diminished litter size and perinatal viability but no malformations were observed. Animal model studies of the teratogenicity of WEE virus are not known. Therefore, WEE virus was selected for study. Since the teratogenicity of the above three viruses is of considerable concern and since, as reported by London et al. (’751, the rhesus monkey is apparently a good primate model for viral teratology studies, an examination of the effects of influenza, mumps and WEE viruses in fetal rhesus monkeys was initiated. Specifically, results from intracerebral inoculations of fetuses and of inoculations of virus into the amniotic sac with attempts a t viral recovery from fetuses are reported. STUDY DESIGN
Two experiments are reported herein. The first, “Virus Transmission,” involved individual placement of each of the three viruses into the amniotic sacs of pregnant rhesus monkeys. The second, “Viral Teratology,” involved
individual placement of each of the three viruses and normal saline (as controls) intracerebrally into fetal rhesus monkeys. In the virus transmission study a total of 26 pregnant rhesus monkeys (Macaca mulatta) with no detectable antibody for influenza or mumps virus or with less than 1:64 WEE antibody titer were inoculated (only 5 of 119 females examined were WEE negative, thus it was not possible to obtain an adequate number of WEE antibody-free animals). Since Kilham and Margolis (’75) reported that hamsters developed hydrocephalus after inoculation of mumps virus into the amniotic sac, we attempted to determine in the nonhuman primate if virus placed in the amniotic sac would infect the fetus and replicate in the fetal tissues. Nine animals received influenza virus, nine received WEE virus and eight received mumps virus. In each virus group two or three animals aborted within 72 hours following inoculation. Since these abortions were believed to have resulted from surgical or manipulative trauma, they were excluded from the study, thus, a total of 18 animals, six in each virus group were studied. Inoculations were made on the ninetieth gestational day (165 days is the average gestation period) and caesarean section deliveries of t h e fetuses were performed on either the ninety-sixth or ninety-seventh gestational day. In experiment 11, the viral teratology study, a total of 13 pregnant rhesus monkeys with no detectable antibody for influenza or mumps virus or less than 1:64 titer for WEE virus were inoculated. London et al. (’75) utilized the intracerebral inoculation route and reported malformations in rhesus monkey fetuses from influenza virus. Thus, these teratology studies were designed to determine if virus placed intracerebrally into fetuses would replicate in the central nervous system and, if so, what teratogenic effects, if any, may be exerted. In the influenza and mumps virus groups four animals were inoculated with each virus, three received WEE virus and two were inoculated with sterile normal saline as controls. One animal in the influenza and one in the mumps group aborted within 48 hours and were excluded because the abortion was probably due to surgical or manipulative trauma. Thus, three animals in each of the virus groups and two in the saline control group were studied. Inoculations were made on the ninetieth gestational day and caesarean deliv-
MONKEY FETAL MALFORMATIONS FROM MUMPS, WEE AND "FLU'
eries were performed on the one-hundred fifty-eighth gestational day. Breeding
Breeding was accomplished in outdoor pens via harem structure with date of conception established by thrice weekly swabbing of the vagina for observation of menstrual flow coupled with examination of the uterus every seven days by means of recto-abdominal digital palpation. Upon diagnosis of a pregnancy the menstrual cycle chart was consulted and conception calculated to have occurred 12 days after cessation of menstrual flow. In a few instances in which pregnancy occurred without a preceding menstrual flow, the conception date was estimated based on failure to detect uterine enlargement seven days prior and on the basis of size and turgidity of the gravid uterus at the time pregnancy was determined. The conception date, thus established, was considered day one of gestation. Gravid females were removed from t h e harems and housed individually (or in a few instances 2 per cage) in indoor suspended large primate cages until the date of virus inoculation. Following inoculation pregnant monkeys were housed individually in cages and held for at least 28 days in cubicle isolation units inside a two corridor isolation suite of rooms. Prenatal observations included daily assessment of stool characteristics, appetite, vaginal bleeding and general appearance. In the week prior to date of inoculation the uterus of each female was palpated to evaluate the status of the pregnancy and fetal viability. Inoculation of fetuses
The pregnant monkeys were given ketamine and atropine as a preanesthetic. After orotracheal intubation, animals were anesthetized with halothane-oxygen mixture. In the virus transmission study a paramedian laparotomy was performed and with one hand underneath, the gravid uterus was elevated and, exercising caution to avoid penetrating the placenta, a 27-gauge needle was inserted through the myometrium into the amniotic sac. A volume of 0.25 ml of amniotic fluid was removed and with the needle remaining in place the syringe was removed and discarded and replaced by a syringe containing 0.25 ml of virus inoculum which was injected. Additional amniotic fluid was then drawn into the syringe and expelled into the amniotic sac to
55
insure delivery of the entire viral inoculum. The needle was then withdrawn, the uterus returned to normal position and the laparotomy closed by routinely suturing peritoneum, muscle sheaths and skin. In the viral teratology study the surgical exposure of the uterus was the same. The inoculation was made after palpating the fetal head and maneuvering it into a position so that penetration of the placenta was avoided. A 27gauge needle was inserted through the intact myometrial wall and through the fetal anterior fontanelle; and 0.25 ml of virus suspension or, in the case of controls, a similar volume of normal saline was deposited into the brain. The needle was withdrawn, and the surgery completed as described above. Virus inoculum Hong Kong influenza virus (A2IAichil2168) obtained from the National Institute for Neurological, Communicative Disorders and Stroke (NINCDS) as infected allantoic fluid from the ninth embryonated egg passage and containing l o 4 median egg infectious doses (EIDSo)per 0.2 ml was stored in 1ml aliquots a t - 80°C until immediately before use. Mumps virus strain P, No. 24 obtained from NINCDS was grown in African Green Monkey Kidney line (Vero) cells and frozen a t - 80°C as 2 ml aliquots containing lo5O median tissue culture infectious doses (TCIDSo)per 0.1 ml until immediately before use. WEE virus of the vaccine strain P, Clone 15 was obtained from NINCDS in sealed glass ampoules, frozen a t -8O"C, and contained lo6 plaque-forming units (PFU) per 0.1 ml according to titrations performed in chick embryo fibroblasts. This virus stock was found t o contain lo4O TCIDS0units per 0.1 ml upon titration in Vero cells. It was stored at - 80°C until immediately before use. Harvest of fetal preparations
Six or seven days post inoculation (ninetysixth or ninety-seventh day of gestation) fetuses in the virus transmission study were delivered via caesarean section, umbilical cord blood samples were collected and fetuses were killed immediately by exsanguination. Attempts at viral isolation were made from amniotic fluid, placenta, fetal throat, cerebrum, cerebellum, medulla, spinal cord, lung, heart, thymus, liver, spleen, kidney, eye and the dam's throat and HI titers were performed on fetal blood.
56
MORELAND, GASKIN, SCHIMPFF, WOODARD AND OLSON
enza virus. Doubling dilutions of the fluids in dextrose-gelatin-Verona1 solution (DGV) were made in U-bottomed microtiter plates and an equal volume of 0.5%washed chicken erythrocytes was added to each well. After incubation a t 4OC for approximately 30 to 40 minutes, the plates were scrutinized for evidence of hemagglutination. Vero cell cultures inoculated with specimens (swab samples, tissue suspensions, cerebrospinal fluid, etc.) in order to recover WEE or mumps viruses were observed regularly over five to seven days for the development of CPE. In the absence of CPE, culture fluids were decanted and monolayers treated with 1.0 ml of trypsin-EdTA solution in order to mono-disperse the cells. Four (4.0) milliliters of growth medium (Hank’s MEM glu10%FBS) were added to each tube, tamine thereby effecting a 1:5 split of the cells in each inoculated culture tube. These cultures Virus isolation were observed regularly over seven additional Throat swab, cerebrospinal fluid, amniotic days for the development of CPE. Direct primary cultures of lung and kidney fluid samples and 10%suspensions of fetal and placental tissues (in Hank’s minimal essential were made by trypsinization from all fresh medium [MEMI containing 10% fetal bovine fetuses inoculated with mumps or WEE serum [FBSI) were inoculated into primary viruses. These cultures were carefully scrutiAfrican green monkey kidney cells (AGMK, nized for the development of CPE. After seven Flow Laboratories, Rockville, Maryland) in days or upon reaching confluency, t h e monoattempts to recover influenza virus. Attempts layers were dispersed with trypsin-EdTA soluto recover mumps and WEE viruses were tion, split 1:2-1:4, and the resulting monomade in the same fashion by inoculation of layers were observed over another five to Vero cells. In each case two to four confluent seven days for the presence of CPE. monolayers in culture tubes were challenged Serological studies with 0.2 ml of inoculum after decanting of the cell culture medium. After incubation a t 37°C Serum samples were obtained from each for one hour, the tubes were rinsed with Dul- female rhesus a t the time of acquisition and a t becco’s phosphate buffered saline (PBS), re- the time of Caesarian section. The presence of plenished with maintenance medium, and ob- antibody to mumps, WEE, or influenza viruses served regularly for the development of cyto- was determined by microtiter hemagglutipathic effects. nation-inhibition tests in accordance with Tubes of primary AGMK cells inoculated for standard procedures as described by Henle (‘69),Hammon and Sather (‘69) and Robinson influenza virus recovery were incubated a t 35°C. They were maintained with MEM with- and Dowdle (’69).Mumps HA antigen was obout fetal bovine serum supplementation in tained commercially as frozen infected egg order to minimize the effect of non-specific in- fluids. WEE HA antigen was supplied by the hibiting substances in serum as described by Center for Disease Control (Atlanta, Georgia) Robinson and Dowdle (’69).After daily obser- as lyophilized infected suckling mouse brain vation for seven days, 0.2 ml of the fluids of extracts. Influenza HA antigen was supplied cell cultures failing to show cytopathic effects by NINCDS as frozen infected allantoic fluid (CPE) were inoculated into fresh AGMK pri- from embryonated eggs. Cord blood samples obtained a t the time of mary cell cultures which were then observed for an additional seven days. All cell culture Caesarean section were also examined for the fluids from animals receiving influenza virus presence of HI antibodies. were tested for their ability to hemagglutiiFlow Laboratories, P. 0. Box 22C, 1710 Chapman Avenue, nate chicken erythrocytes as a further indica- Rockville, Maryland 20852. Microbiological Associates, Inc., 4733 tion of whether or not they contained influ- Bethesda Avenue, Bethesda, Maryland 20014.
In the viral teratology study gestation was allowed to proceed until t h e one-hundred fifty-eighth day before delivery of fetuses via caesarean section. Umbilical cord blood samples were collected and fetuses were killed immediately with sodium pentobarbital. For each virus the whole brains from two fetuses were obtained for histopathological studies and the brain from the remaining fetus was examined immediately for gross signs of malformations. Block sections of cerebellum, cerebrum, and medulla were then obtained for fluorescent antibody (FA) and virus isolation studies. Specimens for FA and virus isolation studies were also obtained from other organs of all fetuses and included spinal cord (thoracic), lung, heart, thymus, liver, spleen, kidney, and eyes. Amniotic fluid, fetal throat and maternal throat were also sampled for virus.
+
+
MONKEY FETAL MALFORMATIONS FROM MUMPS, WEE A N D “FLU’
Fluorescent antibody studies (FA) FA studies were planned and appropriate specimens were collected, however, as of this writing technical problems have prevented a conclusive determination on the significance of immunofluorescence studies.
Pathology All fetuses were examined for malformations externally by visual inspection and internal organs were also visually inspected. However, since our principal suspicion was that these viruses would affect the central nervous system (CNS) detailed attention was given to the CNS. Brains were removed with meninges intact. After external examination, they were placed in 10%neutral buffered formalin for ten days prior to further evaluation. Nine 0.5-cm coronal sections of the cerebral hemispheres were made after removal of the brainstem and cerebellum by transverse section just rostra1 to the midbrain. All sections were examined with a dissecting microscope as well as gross observation with particular attention to the ventricular system and the ependymal lining for evidence of inflammatory changes. After photography, specimens were obtained for histologic evaluation from the following areas: A. Transverse section of mid-medulla; B. Transverse section of pons; C. Transverse section of midbrain with aqueduct of Sylvius; D. Midline superior cerebellar vermis; E. Hippocampus; F. Lenticular nucleus; G. Mid-thalamus and third ventricle; H. Lateral ventricle; I. Choroid plexus; J. Cerebral cortex. The blocks were paraffin embedded and sectioned for histology. The sections were stained with hematoxylin and eosin and further stains of selected blocks were done with phosphotungstic acid hematoxylin, Holzer, luxol fast blue and a silver axonal stain. RESULTS
Virus transmission study Fetal infections were demonstrated in all fetuses in the case of all three viruses (table 1). Influenza virus was recovered only from tissues in contact with amniotic fluid such as fetal throat, lung, and eyes. Virus also was recovered from amniotic fluid and placenta. Mumps virus was recovered from fetal throat, placenta, amniotic fluid, cerebrum, cerebellum, medulla, spinal cord, lung, heart, thymus, liver, spleen, kidney, and eyes. WEE virus was recovered from placenta, amniocic
57
fluid, eyes, lung, cerebellum, medulla, and spinal cord. Serological data were variable (table 1). In general, maternal antibody titers remained the same or showed slight increases, although two animals receiving WEE virus had a decline in titer. Due to oversight or technical difficulties, umbilical cord blood samples were not available on all fetuses. However, of two samples from the influenza group, one was negative and one 1:8; of five samples from the mumps group, three were negative and two 1:4; of five samples from the WEE group, two were negative, two 1:4 and one 1:8.
Viral teratology study (table 2) The three fetuses exposed to influenza virus were anatomically normal on gross and histologic examination. No post infection fetal mortality was observed. One umbilical cord blood sample was obtained which revealed an HI titer of 1:512 while the maternal titer remained unchanged a t 1:4.Two other maternal blood samples showed increasing titers. Of the three fetuses exposed to mumps virus only one lived to reach the one hundred fiftyeighth gestational day. Two fetuses were aborted on the one hundred forty-third and one hundred forty-ninth gestational days. Both were in a poor state of preservation and development of these fetuses appeared to have been arrested two or three weeks prior to the abortion. Apparently they had been retained in utero, even though dead, for the period since development ceased. Tissues were too necrotic for histology. One umbilical cord blood sample was obtained which revealed an HI titer of 1:8 while the maternal titer changed from negative to 1:4. The remaining two mothers aborted and blood samples were not obtained. The three fetuses exposed to WEE virus exhibited malformations. One fetus was aborted on the one hundred forty-seventh gestational day and, although partially decomposed a t birth, histologic examination of brain was accomplished. Of the remaining fetuses one was born spontaneously in the early morning of the one hundred fifty-eighth gestational day and the other was delivered by Caesarean section a t 158 days. Umbilical cord blood of the two fetuses sampled were 1:1024 and 1:256. The dams also showed significant increase in titers. Gross observations All three WEE virus specimens showed
R
Q
0 P
N
M
0
-
-
-
-
-
Throat dam
5
+ + + +
Throat fetus
-
+ +
5
+
+
+
Placenta
3
+
Amniotic fluid
' Not done due to oversight or technical difficulties.
Total XI6
WEE
Total X/6
L
K
J
F Total XI6 Mumps G H I
D E
C
B
Influenza A
Animal
4
+
+ + +
Cerebrum
4
+
+ + +-
Cerebellum
~
6
+ + ++ + +
+
+
+ + + +
+ ++ + 4
~
~
3
+ ++
-
0
-
-
-
3
+ + +
-
0
-
-
-
-
2
-
+ +
0
-
-
-
-
Lung Heart Thymus Liver
6
~~
0
-
-
-
Spinal cord
3
-
+ + + +
0
-
-
-
Spleen
_
-
Summary of virus recoueries and HI titers-transmission study
TABLE 1
3
+
+
-
-
+
-
0
-
-
-
ffidney
5
+
+
+-
+
2
-
-
+
+
Eye
_ ~
~
_
1 1 1 6
1
1 1
16
1
1 1
1
1
X/6
Total infected
1:4 1:4 Neg 1.4 1:4 Neg
1:4 Neg Neg Neg Neg Neg
Pre inw titer dam
~
1:8 1:8 1:8 1:8 1:8 ND '
1:4 ND ' 1:4 1:4 1:4 1:8
Titer a t C-section dam
1:8
ND I ND I ND ' 1:8 Negl ND
Umb cord blood titer
~
E m
59
MONKEY FETAL MALFORMATIONS FROM MUMPS, WEE AND “FLU’
gross evidence of severe hydrocephalus. The gyral pattern was obliterated over the occipital-parietal-temporal region in two of the three cases with marked thinning of the gyri apparent at the fronto-parietal junction. The only normal gyral formation appeared in the region of the frontal and pre-frontal cortex. In the two most severe cases the thinned cortex was translucent. The most extreme ventricular enlargement occurred in the occipital and temporal regions of the lateral ventricles (figs. 1, 2). The enlarged third ventricles showed communication with an enlarged
aqueduct of Sylvius rostrally which, in the two cases which could be examined, showed stenosis and occlusion a t the rostra1 level of midbrain (fig. 3). In this peri-aqueductal region, necrosis, gliosis and calcification could be seen by use of the dissecting microscope. The foramina Luschka and Magendie were patent in the two cases of complete examination. Microscopic observations
Microscopic examination of all cases showed similar findings. Although a mild meningeal
TABLE 2
Summary of malformations- viral teratology study Virus Gross
Influenza Influenza Influenza Mumps
Mumps Mumps
WEE
WEE
WEE
Saline Saline
Microscopic
Pre-inoc titer-dam
Titer at C-section dam
Umh. blood
This was apparent full term infant delivered spontaneously, normal Normal-C Section Normal-C Section
Brain for FA
Neg
1:512
ND’
No pathologic changes No pathologic changes
Neg 1:4
1:8 1:4
ND2 1:512
Normal in gross appearance. Aborted after 59 days. No hair present. Believed dead for several days or weeks Delivered by C section. Brain for FA. Normal Aborted after 53 days. Fetus recovered. Dead for several days or weeks. Cortical flattening, severe post mortem decomposition
Aborted. Too necrotic
Neg
ND1
ND’
Brain for FA
Neg
1:4
1%
Aborted. Too necrotic
1:4
ND2
ND1
Delivered by C Section. Loss of gyral pattern due to thinning over the occipital, parietal and temporal cortex. Hydrocephalus, aqueduct of Sylvius occluded, superior to fourth ventricle, fourth ventricle patent but small Delivered by C Section. Fetus recovered. Loss of gyral pattern due to thinning over the occipital, parietal and temporal cortex. Hydrocephalus, aqueduct of Sylvius occluded, post mortem decomposition Spontaneous birth. Loss of gyral pattern over the occipital lobe and thinning of cortex of temporal and parietal lobes. Hydrocephalus noted
Hydrocephalus severe in lateral and third ventricle, encephalitis with necrosis, dystrophic calcification, ependymitis, severe mononuclear infiltrate, chronic meningeal inflammation
Neg
1:128
1:1024
Hydrocephalus severe in lateral and third ventricle, encephalitis chronic meningeal inflammation, ventriculitis, calcification, thinned cortex
1:4
ND2
ND2
Half brain for FA thus allowing only limited histology. Hydrocephalus slightly leas severe. Slight enlargement of fourth ventricle. Perivascular cuffing in brain stem, foci of chronic inflammation in brain stem, focal ependymal denudation without inflammation in lateral venticles
1:16
1:128
1:2563
Normal C Section Normal C Section
No pathologic changes No pathologic changes
’ Not done because of abortion or spontaneous delivery. ‘Not done due to oversight or technical difficulty. Fetal heart hlmd sample.
Cord titer
60
MORELAND, GASKIN, SCHIMPFF, WOODARD AND OLSON
Fig. 1 Lateral view of the brain from a WEE infected fetus delivered on the one hundred fifty-eighth gestational day. The cerebral cortex is collapsing inwardly as a result of severe hydrocephalus.
Fig. 2 Coronal sections demonstrate the marked thinning of cerebral cortex associated with massive ventricular enlargement.
MONKEY FETAL MALFORMATIONS FROM MUMPS, WEE AND “FLU”
61
Fig. 3 Sections of midbrain at the same level from a hydrocephalic WEE injected fetus (left) and a normal fetus of identical gestational age (right). The severe periaqueductal and midbrain encephalitis has resulted in distortion and shrinkage of the infected tissue. The rostral portion of the aqueduct of Sylvius is dilated and leads to a blind pouch with complete occlusion just rostral to the fourth ventricle. Adjacent to the aqueduct the shrunken tissue has a whitish, granular appearance as a result of dystrophic calcification and gliosis.
mononuclear reaction was seen, the ependymal findings were marked. Much of the ependymal surface was denuded and replaced by mononuclear inflammatory cells and fibrous astrocytosis. Marked thinning of the cerebral cortex with lymphocyte-cuffed vessels and compression of the subcortical white matter were prominent (fig. 4). In the occipito-parietal cortex, there was only a very thin layer of neuronal cells and a gliotic molecular layer. In the sections through the aqueduct of Sylvius and rostral fourth ventricle, severe perivascular cuffing with lymphocytes and distended macrophages were prominent (fig. 5). Gliosis was observed in the quadrigeminal plate and sporadically throughout the tectal and pre-tectal areas in more caudal portions of the brainstem. Dystrophic calcification adjacent to the damaged ependymal lining was present in the periaqueductal region. Focal ependymal denudation, gliosis, periventricular inflammation, and patchy necrosis occurred throughout the brainstem adjacent to the fourth ventricle. The cerebellum showed only pre-myelination gliosis without evidence of encephalitis.
In summary, these findings indicate severe encephalitis affecting all portions of the brain except cerebellum with widespread ependymal damage, periependymal gliosis and aqueductal stenosis occurring in the midbrain region. Hydrocephalus as a result of this encephalitic process appears to be a t least partially attributable to the aqueductal stenosis. DISCUSSION
Influenza virus This report confirms reports of Fuccillo and Sever (‘73),Johnson and Johnson (‘69)and London et al. (‘75) that infections with influenza virus are established in fetal animals following experimental exposure. Virus was not recovered from organs other than those in contact with infected amniotic fluid. Ruben et al. (‘75) indicate that the virus has been isolated from lungs of abortuses of women with severe influenza infections. The portal of entry in post-natal human natural infections is known to be via cells of the respiratory tract. Therefore, this result is in concurrence. Because the virus has not spread to other organ tissues, these results are believed
62
MORELAND, GASKIN, SCHIMPFF, WOODARD AND OLSON
Fig. 4 The thinned cerebral cortical mantle is composed of compressed gray and white matter with perivascular lymphocytic cuffing, gliotic fibrous astrocytes and an inner denuded ependymal surface. Along the ventricular surface, inflammatory and glial cells form a multi-layered periventricular lining. x 100.
Fig. 5 The aqueduct of Sylvius and rostral fourth ventricle have the most severe inflammatory changes. Perivascular lymphocytic cuffing, distended macrophages, dystrophic calcification and intense gliosis are shown in this section immediately adjacent to the occluded aqueduct. x 100.
MONKEY FETAL MALFORMATIONS FROM MUMPS, WEE AND “FLU’
63
erally considered evidence that a viral agent has been present and an immunologic reaction has been evoked. Since antibody was not uniformly present in all fetuses from which virus was isolated we conclude that the elapsed time between infection and sample time was insufficient (7 days only) for detectable antibody production or that some fetuses were incapable of producing antibody at this stage of development (90-97 days gestation). The very low titers noted in most of the fetuses probably indicate that not enough time has elapsed Mumps virus for antibody production. Results confirm experimental fetal infecFA studies for detection of viral antigens in tions in hamsters as reported by Kilham and tissues were planned but as of this writing Margolis (‘75) and in rhesus monkeys as re- technical problems have prevented a conported by St. Geme and Van Pelt (‘74) and clusive determination on the significance of London et al. (’71). Further, the evidence pre- these studies. sented herein that in the primate fetus In attempting to limit the teratology experimumps virus invades essentially all organ tis- ments to the intracerebral inoculations of sues is a significant finding. This is true par- three animals with each virus (because of ticularly since it more closely mimics mumps scarcity, humane considerations and high cost infection in the human and since Johnson of nonhuman primates) it was realized that (‘68b) has specifically shown that in the ham- the sample size was marginal. Nevertheless, ster the virus does not seem to replicate in the data clearly point to a lack of teratogenic non-neural tissue. This assumes even greater effect of influenza virus while dramatically importance in light of St. Geme and Van Pelt’s showing fetal mortality and teratogenesis re(‘72) demonstration that virus could be re- sulting from mumps and WEE virus. covered from triturated fetal tissues of rhesus Mumps infection resulted in death of two of monkeys following intravenous inoculation of three fetuses. This number is insufficient to the dam, thus confirming transplacental in- draw firm conclusions and simply suggests fection. This suggests that the primate should additional studies to elucidate teratogenesis be more intensively studied as an animal and pathogenesis while supporting previous model which will simulate mumps infection a t observations that this virus results in a high incidence of fetal mortality. critical phases of development. The teratology study supports the observaThe 100%incidence of hydrocephalus in the tions of London et al. (‘71) that a high rate of three fetuses receiving WEE virus is striking, fetal mortality occurs in experimental infec- however, and strongly suggests the signifition in rhesus monkeys and of Siege1 et al. cant teratogenic potential of this virus. Although resulting in aqueductal stenosis, (‘66) that fetal mortality is increased in the pathology demonstrated in these fetal prihumans following natural mumps infection. mate specimens infected with WEE should not W E E virus be confused with noninflammatory aqueducThis is the first report of an experimental tal stenosis first reported by Johnson (‘68a) as animal study relating to fetal infections and a results of mumps inoculation in suckling clearly shows that (1) WEE virus infection hamsters. In those experiments, infection has can be established in fetal primates and (2) been shown to be almost exclusively limited to infection of fetuses with WEE virus may pro- ependymal cells and choroid plexus. Subseduce a severe encephalitis resulting in hydro- quent loss of damaged ependymal cells into cephalus. Considering the fact that infection the aqueduct and the associated reaction rewith this virus in adult humans is often sub- sults in obstructive hydrocephalus. No spread clinical these findings become increasingly of the mumps virus to the brain parenchyma significant and t h e implications assume was demonstrated nor were histologic findings suggestive of generalized encephalitis. The reserious proportions. sults described here demonstrate widespread General inflammatory histologic changes throughout The presence of antibodies in serum is gen- the central nervous system parenchymal tis-
to support the indications that fetal infection with influenza viruses does not lead to development of congenital malformations. This latter conclusion was further supported by results of the teratology experiment in which no malformations nor fetal deaths occurred following intracerebral instillation into the fetal brain. These results failed to confirm the observations of London et al. (’75) that intracerebral inoculation of influenza A virus caused hydrocephalus.
64
MORELAND, GASKIN, SCHIMPFF, WOODARD AND OLSON
sue indicative of severe encephalitis. In addition, ependymal denudation, periventricular inflammation and subsequent aqueductal stenosis resulted in severe obstructive hydrocephalus. It is not known if alterations in dosage, rout of inoculation, age of fetus a t exposure, or previous maternal immunity might restrict WEE infection to ependymal cells, thereby resulting in obstructive aqueductal stenosis not accompanied by generalized encephalitis. Based on the growing body of information which strongly suggests that obstructive hydrocephalus may be the result of early fetal ependymal infection, specific high risk infections need to be identified and methods devised to reduce fetal morbidity. This identification of WEE virus as a significant teratogenic agent has added to the growing number of experimentally proven teratogenic viral infections which require further evaluation; i t is particularly important because of reports of transplacental WEE human fetal infections. ACKNOWLEDGMENTS
The authors wish to acknowledge the advice and direction given by Doctor William T. London of the National Institute of Neurological, Communicative Disorders and Stroke who served as the Project officer for the contract. Also, we acknowledge the invaluable technical assistance of Marie Powell and Glenda Hall. LITERATURE CITED Cohen, R., R. E. O'Connor, T. E. Townsend, P. A. Webb and R. W. McKey 1953 Western equine encephalomyelitis: Clinical observations in infants and children. J. Pediatrics, 43: 26-34. Coops, S. C., and L. E. Giddings 1959 Transplacental transmission of Western equine encephalitis. Report of a case. Pediatrics, 24: 31-33. Ennis, F. A., H. E. Hopps, R. D. Douglas and H. M. Meyer 1969 Hydrocephalus in hamsters: Induction by natural attenuated mumps virus. J. Infect. Dis., 119: 75-79. Fuccillo, D. A., and J. L.Sever 1973 Viral teratology. Bacteriol. Rev., 37: 19-31. Hamburger, V., and K.Habel 1947 Teratogenic and lethal
effects of influenza A and mumps viruses on early chick embryos. Proc. SOC. Exp. Biol., 66: 608-617. Hammon, W. M., and G.E. Sather 1969 Arboviruses. In: Diagnostic Procedures for Viral and Rickettsial Infections. E. H. Lennette and N. J. Schmidt, eds. American Public Health Association, Inc., New York, pp. 227-280. Henle, W. 1969 Mumps virus. In: Diagnostic Procedures for Viral and Rickettsial Infections. E. H. Lennette and N. J . Schmidt, eds. American Public Health Association, Inc., New York, pp. 457-482. Johnson, R. E., and K. P.Johnson 1969 Hydrocephalus as a sequella of experimental myxovirus infections. Exp. Molecular Path., 20: 68. Johnson, R. T. 1968a Hydrocephalus following viral infection: The pathology of aqueductal stenosis developing after experimental mumps virus infection. J. Neuropathol. Exp. Neurol., 27: 591-606. 1968b Mumps virus encephalitis in the hamster: Studies of the inflammatory response and noncytopathic infection of neurons. J. Neuropath. and Experiment. Neurol., 27: 80-95. Kilham, L., and G. Margolis 1975 Induction of congenital hydrocephalus in hamsters with attenuated and natural strains of mumps virus. J. Infect. Dis., 132: 462-466. London, W. T., B. Curfman and J. L. Sever 1971 Mumps infection of t h e rhesus monkey fetus. Teratology, 4: 234 (Abstract). London, W. T., D. A. Fuccillo, J. L. Sever and S. G. Kent 1975 Influenza virus as a teratogen in rhesus monkeys. Nature, 255: 483-484. MacKenzie, J. S.,and M. Houghton 1974 Influenza infections during pregnancy: Association with congenital malformations and with subsequent neoplasms in children, and potential hazards of live virus vaccines. Bacteriol. Rev., 38: 356-370. Medovy, H. 1943 Western equine encephalomyelitis in infants. J. Pediatrics, 22: 308-318. Robinson, R. O.,and W. R. Dowdle 1969 Influenza viruses. In: Diagnostic Procedures for Viral and Rickettsial Infections. E. H. Lennette and N. J. Schmidt, eds. American Public Health Association Inc., New York, pp. 414-433. Ruben, F. L.,A. Winkelstein and R. E. Sabbagha 1975 In utero sensitization with influenza virus in man. Proc. Soc. Exp. Biol.Med., 149: 881-883. Shinefield, H. R., and T. E. Townsend 1953 Transplacental transmission of western equine encephalomyelitis. J. Pediatrics, 43; 21-25. Siegel, M., H. T. Fuerst and N. S. Peress 1966 Comparative fetal mortality in maternal virus diseases. N. Eng. J. Med., 274: 768-771. Spertzel, R. O.,C. L. Crabbs and R. E. Vaughn 1972 Transplacental transmission of Venezuelan equine e n cephalomyelitis virus in mice. Infect. Immun., 6: 339-343. St. Geme, J. W. Jr., and L. F. Van Pelt 1974 Fetal and postnatal growth retardation association with gestational mumps virus infection of the rhesus monkey. Lab. Animal Sci., 24: 895-899.
ABSTRACT Pregnant Rhesus monkeys were infected via instillation of influenza, mumps and western equine encephalomyelitis viruses respectively into the amniotic sacs a t approximately 90 days gestation to determine if fetal infections would occur. Virus was recovered from fetal tissues after seven days in 100%of the exposed animals. Thus, the viruses are capable of causing fetal infection. Rhesus monkey fetuses were inoculated with influenza, mumps and WEE viruses by the direct intracerebral route a t approximately 90 days gestation to determine possible teratogenicity of the viruses. Influenza virus caused no malformations or measurable fetal effects. Mumps virus resulted in significant fetal mortality, WEE virus resulted in a 100%incidence of encephalitis and hydrocephalus. Thus,mumps and WEE viruses are teratogens in primates and are potential teratogens of man. Numerous viruses are known to seriously and either congenital malformations or subseaffect neonatal development and the effects of quent neoplasms during childhood.” Perhaps prenatal infections with rubella, herpesvirus the strongest evidence supportive of malforsimplex Type I1 and cytomegalovirus are well mations in higher mammalian species to date established. I t has been long suspected, how- resulted from a study by London et al. (‘75) ever, that congenital malformations and fetal which demonstrated occurrence of hydrocephmortality may be attributable to other pre- alus in fetal rhesus monkeys following intracerebral inoculation of influenza A virus. natal virus infections. The observation that neural tube malforma- It is against this background of conflicting retions were induced in chick embryos inocu- ports that we decided to conduct the present lated with influenza virus was first described studies on influenza virus. by Hamburger and Habel (‘47)and has been Siege1 et al. (‘66) reported that human subsequently confirmed by numerous others. maternal mumps virus infection during the Fetal wastage and abnormalities of the ner- first trimester was associated with increased vous system in mice have been reported by fetal mortality, however, infection of fetuses Fuccillo and Sever (‘73) and hydrocephalus was not demonstrated by virus isolation. has resulted from direct intracerebral inocu- Johnson (’68a) reported hydrocephalus in lation of influenza virus into suckling ham- suckling hamsters following intracerebral insters and mice as reported by Johnson and oculation with mumps virus and Ennis et al. Johnson (‘69). Evidence incriminating influ- (’69) confirmed this work. Kilham and Marenza as a human teratogen and as a predis- golis (‘75)reported induction of hydrocephposing factor for neoplasms has been recently alus in hamsters after inoculation of mumps reviewed by MacKenzie and Houghton (’74). virus into the amniotic sac and St. Geme and While many studies have demonstrated a Van Pelt (’74)described infections in fetal significant relationship, perhaps equally as Received April 27, ‘78. Accepted Jan. 20, ’79. many have not, thus leading them to the conSupported by Contract NS-5-2318from the National Institutes clusion that “probably no direct association of Health. U. S. Public Health Service, Department of Health. exists between maternal influenza infections Education and Welfare. 1
TERATOLOGY(1979) 20: 53-64.
53
54
MORELAND, GASKIN, SCHIMPFF. WOODARD AND OLSON
rhesus monkeys following intravenous inoculation of the mother thus indicating transplacental passage of the virus, London et al. (’71) reported a high rate of fetal mortality in rhesus monkey fetuses inoculated intracerebrally. No malformations occurred in either of the latter two studies, although both reports indicated growth retardation in fetal animals and St. Geme and Van Pelt further observed this in neonates. Thus, there appears to be a clear association between mumps virus and a high rate of fetal mortality. The relationship to congenital malformations is less certain although strongly suggestive. For this reason mumps virus was included in the present study. Probable transplacental infections of human fetuses with western equine encephalitis (WEE) virus have been reported by Medovy (’431, Shinefield and Townsend (’531,Cohen et al. (’53), and Copps and Giddings (’59), however, all were late in gestation (10-12 days prior to parturition) and malformations were not observed. Nevertheless, the potential for infection of mothers early in gestation is great and since WEE virus infections in adult humans are often mild, transient, brief infections which often go unrecognized the probability of this togavirus contributing to the incidence of malformations in humans is of considerable interest and concern. Spertzel et al. (’72) showed transplacental passage of Venezuelan equine encephalitis virus in mice which resulted in diminished litter size and perinatal viability but no malformations were observed. Animal model studies of the teratogenicity of WEE virus are not known. Therefore, WEE virus was selected for study. Since the teratogenicity of the above three viruses is of considerable concern and since, as reported by London et al. (’751, the rhesus monkey is apparently a good primate model for viral teratology studies, an examination of the effects of influenza, mumps and WEE viruses in fetal rhesus monkeys was initiated. Specifically, results from intracerebral inoculations of fetuses and of inoculations of virus into the amniotic sac with attempts a t viral recovery from fetuses are reported. STUDY DESIGN
Two experiments are reported herein. The first, “Virus Transmission,” involved individual placement of each of the three viruses into the amniotic sacs of pregnant rhesus monkeys. The second, “Viral Teratology,” involved
individual placement of each of the three viruses and normal saline (as controls) intracerebrally into fetal rhesus monkeys. In the virus transmission study a total of 26 pregnant rhesus monkeys (Macaca mulatta) with no detectable antibody for influenza or mumps virus or with less than 1:64 WEE antibody titer were inoculated (only 5 of 119 females examined were WEE negative, thus it was not possible to obtain an adequate number of WEE antibody-free animals). Since Kilham and Margolis (’75) reported that hamsters developed hydrocephalus after inoculation of mumps virus into the amniotic sac, we attempted to determine in the nonhuman primate if virus placed in the amniotic sac would infect the fetus and replicate in the fetal tissues. Nine animals received influenza virus, nine received WEE virus and eight received mumps virus. In each virus group two or three animals aborted within 72 hours following inoculation. Since these abortions were believed to have resulted from surgical or manipulative trauma, they were excluded from the study, thus, a total of 18 animals, six in each virus group were studied. Inoculations were made on the ninetieth gestational day (165 days is the average gestation period) and caesarean section deliveries of t h e fetuses were performed on either the ninety-sixth or ninety-seventh gestational day. In experiment 11, the viral teratology study, a total of 13 pregnant rhesus monkeys with no detectable antibody for influenza or mumps virus or less than 1:64 titer for WEE virus were inoculated. London et al. (’75) utilized the intracerebral inoculation route and reported malformations in rhesus monkey fetuses from influenza virus. Thus, these teratology studies were designed to determine if virus placed intracerebrally into fetuses would replicate in the central nervous system and, if so, what teratogenic effects, if any, may be exerted. In the influenza and mumps virus groups four animals were inoculated with each virus, three received WEE virus and two were inoculated with sterile normal saline as controls. One animal in the influenza and one in the mumps group aborted within 48 hours and were excluded because the abortion was probably due to surgical or manipulative trauma. Thus, three animals in each of the virus groups and two in the saline control group were studied. Inoculations were made on the ninetieth gestational day and caesarean deliv-
MONKEY FETAL MALFORMATIONS FROM MUMPS, WEE AND "FLU'
eries were performed on the one-hundred fifty-eighth gestational day. Breeding
Breeding was accomplished in outdoor pens via harem structure with date of conception established by thrice weekly swabbing of the vagina for observation of menstrual flow coupled with examination of the uterus every seven days by means of recto-abdominal digital palpation. Upon diagnosis of a pregnancy the menstrual cycle chart was consulted and conception calculated to have occurred 12 days after cessation of menstrual flow. In a few instances in which pregnancy occurred without a preceding menstrual flow, the conception date was estimated based on failure to detect uterine enlargement seven days prior and on the basis of size and turgidity of the gravid uterus at the time pregnancy was determined. The conception date, thus established, was considered day one of gestation. Gravid females were removed from t h e harems and housed individually (or in a few instances 2 per cage) in indoor suspended large primate cages until the date of virus inoculation. Following inoculation pregnant monkeys were housed individually in cages and held for at least 28 days in cubicle isolation units inside a two corridor isolation suite of rooms. Prenatal observations included daily assessment of stool characteristics, appetite, vaginal bleeding and general appearance. In the week prior to date of inoculation the uterus of each female was palpated to evaluate the status of the pregnancy and fetal viability. Inoculation of fetuses
The pregnant monkeys were given ketamine and atropine as a preanesthetic. After orotracheal intubation, animals were anesthetized with halothane-oxygen mixture. In the virus transmission study a paramedian laparotomy was performed and with one hand underneath, the gravid uterus was elevated and, exercising caution to avoid penetrating the placenta, a 27-gauge needle was inserted through the myometrium into the amniotic sac. A volume of 0.25 ml of amniotic fluid was removed and with the needle remaining in place the syringe was removed and discarded and replaced by a syringe containing 0.25 ml of virus inoculum which was injected. Additional amniotic fluid was then drawn into the syringe and expelled into the amniotic sac to
55
insure delivery of the entire viral inoculum. The needle was then withdrawn, the uterus returned to normal position and the laparotomy closed by routinely suturing peritoneum, muscle sheaths and skin. In the viral teratology study the surgical exposure of the uterus was the same. The inoculation was made after palpating the fetal head and maneuvering it into a position so that penetration of the placenta was avoided. A 27gauge needle was inserted through the intact myometrial wall and through the fetal anterior fontanelle; and 0.25 ml of virus suspension or, in the case of controls, a similar volume of normal saline was deposited into the brain. The needle was withdrawn, and the surgery completed as described above. Virus inoculum Hong Kong influenza virus (A2IAichil2168) obtained from the National Institute for Neurological, Communicative Disorders and Stroke (NINCDS) as infected allantoic fluid from the ninth embryonated egg passage and containing l o 4 median egg infectious doses (EIDSo)per 0.2 ml was stored in 1ml aliquots a t - 80°C until immediately before use. Mumps virus strain P, No. 24 obtained from NINCDS was grown in African Green Monkey Kidney line (Vero) cells and frozen a t - 80°C as 2 ml aliquots containing lo5O median tissue culture infectious doses (TCIDSo)per 0.1 ml until immediately before use. WEE virus of the vaccine strain P, Clone 15 was obtained from NINCDS in sealed glass ampoules, frozen a t -8O"C, and contained lo6 plaque-forming units (PFU) per 0.1 ml according to titrations performed in chick embryo fibroblasts. This virus stock was found t o contain lo4O TCIDS0units per 0.1 ml upon titration in Vero cells. It was stored at - 80°C until immediately before use. Harvest of fetal preparations
Six or seven days post inoculation (ninetysixth or ninety-seventh day of gestation) fetuses in the virus transmission study were delivered via caesarean section, umbilical cord blood samples were collected and fetuses were killed immediately by exsanguination. Attempts at viral isolation were made from amniotic fluid, placenta, fetal throat, cerebrum, cerebellum, medulla, spinal cord, lung, heart, thymus, liver, spleen, kidney, eye and the dam's throat and HI titers were performed on fetal blood.
56
MORELAND, GASKIN, SCHIMPFF, WOODARD AND OLSON
enza virus. Doubling dilutions of the fluids in dextrose-gelatin-Verona1 solution (DGV) were made in U-bottomed microtiter plates and an equal volume of 0.5%washed chicken erythrocytes was added to each well. After incubation a t 4OC for approximately 30 to 40 minutes, the plates were scrutinized for evidence of hemagglutination. Vero cell cultures inoculated with specimens (swab samples, tissue suspensions, cerebrospinal fluid, etc.) in order to recover WEE or mumps viruses were observed regularly over five to seven days for the development of CPE. In the absence of CPE, culture fluids were decanted and monolayers treated with 1.0 ml of trypsin-EdTA solution in order to mono-disperse the cells. Four (4.0) milliliters of growth medium (Hank’s MEM glu10%FBS) were added to each tube, tamine thereby effecting a 1:5 split of the cells in each inoculated culture tube. These cultures Virus isolation were observed regularly over seven additional Throat swab, cerebrospinal fluid, amniotic days for the development of CPE. Direct primary cultures of lung and kidney fluid samples and 10%suspensions of fetal and placental tissues (in Hank’s minimal essential were made by trypsinization from all fresh medium [MEMI containing 10% fetal bovine fetuses inoculated with mumps or WEE serum [FBSI) were inoculated into primary viruses. These cultures were carefully scrutiAfrican green monkey kidney cells (AGMK, nized for the development of CPE. After seven Flow Laboratories, Rockville, Maryland) in days or upon reaching confluency, t h e monoattempts to recover influenza virus. Attempts layers were dispersed with trypsin-EdTA soluto recover mumps and WEE viruses were tion, split 1:2-1:4, and the resulting monomade in the same fashion by inoculation of layers were observed over another five to Vero cells. In each case two to four confluent seven days for the presence of CPE. monolayers in culture tubes were challenged Serological studies with 0.2 ml of inoculum after decanting of the cell culture medium. After incubation a t 37°C Serum samples were obtained from each for one hour, the tubes were rinsed with Dul- female rhesus a t the time of acquisition and a t becco’s phosphate buffered saline (PBS), re- the time of Caesarian section. The presence of plenished with maintenance medium, and ob- antibody to mumps, WEE, or influenza viruses served regularly for the development of cyto- was determined by microtiter hemagglutipathic effects. nation-inhibition tests in accordance with Tubes of primary AGMK cells inoculated for standard procedures as described by Henle (‘69),Hammon and Sather (‘69) and Robinson influenza virus recovery were incubated a t 35°C. They were maintained with MEM with- and Dowdle (’69).Mumps HA antigen was obout fetal bovine serum supplementation in tained commercially as frozen infected egg order to minimize the effect of non-specific in- fluids. WEE HA antigen was supplied by the hibiting substances in serum as described by Center for Disease Control (Atlanta, Georgia) Robinson and Dowdle (’69).After daily obser- as lyophilized infected suckling mouse brain vation for seven days, 0.2 ml of the fluids of extracts. Influenza HA antigen was supplied cell cultures failing to show cytopathic effects by NINCDS as frozen infected allantoic fluid (CPE) were inoculated into fresh AGMK pri- from embryonated eggs. Cord blood samples obtained a t the time of mary cell cultures which were then observed for an additional seven days. All cell culture Caesarean section were also examined for the fluids from animals receiving influenza virus presence of HI antibodies. were tested for their ability to hemagglutiiFlow Laboratories, P. 0. Box 22C, 1710 Chapman Avenue, nate chicken erythrocytes as a further indica- Rockville, Maryland 20852. Microbiological Associates, Inc., 4733 tion of whether or not they contained influ- Bethesda Avenue, Bethesda, Maryland 20014.
In the viral teratology study gestation was allowed to proceed until t h e one-hundred fifty-eighth day before delivery of fetuses via caesarean section. Umbilical cord blood samples were collected and fetuses were killed immediately with sodium pentobarbital. For each virus the whole brains from two fetuses were obtained for histopathological studies and the brain from the remaining fetus was examined immediately for gross signs of malformations. Block sections of cerebellum, cerebrum, and medulla were then obtained for fluorescent antibody (FA) and virus isolation studies. Specimens for FA and virus isolation studies were also obtained from other organs of all fetuses and included spinal cord (thoracic), lung, heart, thymus, liver, spleen, kidney, and eyes. Amniotic fluid, fetal throat and maternal throat were also sampled for virus.
+
+
MONKEY FETAL MALFORMATIONS FROM MUMPS, WEE A N D “FLU’
Fluorescent antibody studies (FA) FA studies were planned and appropriate specimens were collected, however, as of this writing technical problems have prevented a conclusive determination on the significance of immunofluorescence studies.
Pathology All fetuses were examined for malformations externally by visual inspection and internal organs were also visually inspected. However, since our principal suspicion was that these viruses would affect the central nervous system (CNS) detailed attention was given to the CNS. Brains were removed with meninges intact. After external examination, they were placed in 10%neutral buffered formalin for ten days prior to further evaluation. Nine 0.5-cm coronal sections of the cerebral hemispheres were made after removal of the brainstem and cerebellum by transverse section just rostra1 to the midbrain. All sections were examined with a dissecting microscope as well as gross observation with particular attention to the ventricular system and the ependymal lining for evidence of inflammatory changes. After photography, specimens were obtained for histologic evaluation from the following areas: A. Transverse section of mid-medulla; B. Transverse section of pons; C. Transverse section of midbrain with aqueduct of Sylvius; D. Midline superior cerebellar vermis; E. Hippocampus; F. Lenticular nucleus; G. Mid-thalamus and third ventricle; H. Lateral ventricle; I. Choroid plexus; J. Cerebral cortex. The blocks were paraffin embedded and sectioned for histology. The sections were stained with hematoxylin and eosin and further stains of selected blocks were done with phosphotungstic acid hematoxylin, Holzer, luxol fast blue and a silver axonal stain. RESULTS
Virus transmission study Fetal infections were demonstrated in all fetuses in the case of all three viruses (table 1). Influenza virus was recovered only from tissues in contact with amniotic fluid such as fetal throat, lung, and eyes. Virus also was recovered from amniotic fluid and placenta. Mumps virus was recovered from fetal throat, placenta, amniotic fluid, cerebrum, cerebellum, medulla, spinal cord, lung, heart, thymus, liver, spleen, kidney, and eyes. WEE virus was recovered from placenta, amniocic
57
fluid, eyes, lung, cerebellum, medulla, and spinal cord. Serological data were variable (table 1). In general, maternal antibody titers remained the same or showed slight increases, although two animals receiving WEE virus had a decline in titer. Due to oversight or technical difficulties, umbilical cord blood samples were not available on all fetuses. However, of two samples from the influenza group, one was negative and one 1:8; of five samples from the mumps group, three were negative and two 1:4; of five samples from the WEE group, two were negative, two 1:4 and one 1:8.
Viral teratology study (table 2) The three fetuses exposed to influenza virus were anatomically normal on gross and histologic examination. No post infection fetal mortality was observed. One umbilical cord blood sample was obtained which revealed an HI titer of 1:512 while the maternal titer remained unchanged a t 1:4.Two other maternal blood samples showed increasing titers. Of the three fetuses exposed to mumps virus only one lived to reach the one hundred fiftyeighth gestational day. Two fetuses were aborted on the one hundred forty-third and one hundred forty-ninth gestational days. Both were in a poor state of preservation and development of these fetuses appeared to have been arrested two or three weeks prior to the abortion. Apparently they had been retained in utero, even though dead, for the period since development ceased. Tissues were too necrotic for histology. One umbilical cord blood sample was obtained which revealed an HI titer of 1:8 while the maternal titer changed from negative to 1:4. The remaining two mothers aborted and blood samples were not obtained. The three fetuses exposed to WEE virus exhibited malformations. One fetus was aborted on the one hundred forty-seventh gestational day and, although partially decomposed a t birth, histologic examination of brain was accomplished. Of the remaining fetuses one was born spontaneously in the early morning of the one hundred fifty-eighth gestational day and the other was delivered by Caesarean section a t 158 days. Umbilical cord blood of the two fetuses sampled were 1:1024 and 1:256. The dams also showed significant increase in titers. Gross observations All three WEE virus specimens showed
R
Q
0 P
N
M
0
-
-
-
-
-
Throat dam
5
+ + + +
Throat fetus
-
+ +
5
+
+
+
Placenta
3
+
Amniotic fluid
' Not done due to oversight or technical difficulties.
Total XI6
WEE
Total X/6
L
K
J
F Total XI6 Mumps G H I
D E
C
B
Influenza A
Animal
4
+
+ + +
Cerebrum
4
+
+ + +-
Cerebellum
~
6
+ + ++ + +
+
+
+ + + +
+ ++ + 4
~
~
3
+ ++
-
0
-
-
-
3
+ + +
-
0
-
-
-
-
2
-
+ +
0
-
-
-
-
Lung Heart Thymus Liver
6
~~
0
-
-
-
Spinal cord
3
-
+ + + +
0
-
-
-
Spleen
_
-
Summary of virus recoueries and HI titers-transmission study
TABLE 1
3
+
+
-
-
+
-
0
-
-
-
ffidney
5
+
+
+-
+
2
-
-
+
+
Eye
_ ~
~
_
1 1 1 6
1
1 1
16
1
1 1
1
1
X/6
Total infected
1:4 1:4 Neg 1.4 1:4 Neg
1:4 Neg Neg Neg Neg Neg
Pre inw titer dam
~
1:8 1:8 1:8 1:8 1:8 ND '
1:4 ND ' 1:4 1:4 1:4 1:8
Titer a t C-section dam
1:8
ND I ND I ND ' 1:8 Negl ND
Umb cord blood titer
~
E m
59
MONKEY FETAL MALFORMATIONS FROM MUMPS, WEE AND “FLU’
gross evidence of severe hydrocephalus. The gyral pattern was obliterated over the occipital-parietal-temporal region in two of the three cases with marked thinning of the gyri apparent at the fronto-parietal junction. The only normal gyral formation appeared in the region of the frontal and pre-frontal cortex. In the two most severe cases the thinned cortex was translucent. The most extreme ventricular enlargement occurred in the occipital and temporal regions of the lateral ventricles (figs. 1, 2). The enlarged third ventricles showed communication with an enlarged
aqueduct of Sylvius rostrally which, in the two cases which could be examined, showed stenosis and occlusion a t the rostra1 level of midbrain (fig. 3). In this peri-aqueductal region, necrosis, gliosis and calcification could be seen by use of the dissecting microscope. The foramina Luschka and Magendie were patent in the two cases of complete examination. Microscopic observations
Microscopic examination of all cases showed similar findings. Although a mild meningeal
TABLE 2
Summary of malformations- viral teratology study Virus Gross
Influenza Influenza Influenza Mumps
Mumps Mumps
WEE
WEE
WEE
Saline Saline
Microscopic
Pre-inoc titer-dam
Titer at C-section dam
Umh. blood
This was apparent full term infant delivered spontaneously, normal Normal-C Section Normal-C Section
Brain for FA
Neg
1:512
ND’
No pathologic changes No pathologic changes
Neg 1:4
1:8 1:4
ND2 1:512
Normal in gross appearance. Aborted after 59 days. No hair present. Believed dead for several days or weeks Delivered by C section. Brain for FA. Normal Aborted after 53 days. Fetus recovered. Dead for several days or weeks. Cortical flattening, severe post mortem decomposition
Aborted. Too necrotic
Neg
ND1
ND’
Brain for FA
Neg
1:4
1%
Aborted. Too necrotic
1:4
ND2
ND1
Delivered by C Section. Loss of gyral pattern due to thinning over the occipital, parietal and temporal cortex. Hydrocephalus, aqueduct of Sylvius occluded, superior to fourth ventricle, fourth ventricle patent but small Delivered by C Section. Fetus recovered. Loss of gyral pattern due to thinning over the occipital, parietal and temporal cortex. Hydrocephalus, aqueduct of Sylvius occluded, post mortem decomposition Spontaneous birth. Loss of gyral pattern over the occipital lobe and thinning of cortex of temporal and parietal lobes. Hydrocephalus noted
Hydrocephalus severe in lateral and third ventricle, encephalitis with necrosis, dystrophic calcification, ependymitis, severe mononuclear infiltrate, chronic meningeal inflammation
Neg
1:128
1:1024
Hydrocephalus severe in lateral and third ventricle, encephalitis chronic meningeal inflammation, ventriculitis, calcification, thinned cortex
1:4
ND2
ND2
Half brain for FA thus allowing only limited histology. Hydrocephalus slightly leas severe. Slight enlargement of fourth ventricle. Perivascular cuffing in brain stem, foci of chronic inflammation in brain stem, focal ependymal denudation without inflammation in lateral venticles
1:16
1:128
1:2563
Normal C Section Normal C Section
No pathologic changes No pathologic changes
’ Not done because of abortion or spontaneous delivery. ‘Not done due to oversight or technical difficulty. Fetal heart hlmd sample.
Cord titer
60
MORELAND, GASKIN, SCHIMPFF, WOODARD AND OLSON
Fig. 1 Lateral view of the brain from a WEE infected fetus delivered on the one hundred fifty-eighth gestational day. The cerebral cortex is collapsing inwardly as a result of severe hydrocephalus.
Fig. 2 Coronal sections demonstrate the marked thinning of cerebral cortex associated with massive ventricular enlargement.
MONKEY FETAL MALFORMATIONS FROM MUMPS, WEE AND “FLU”
61
Fig. 3 Sections of midbrain at the same level from a hydrocephalic WEE injected fetus (left) and a normal fetus of identical gestational age (right). The severe periaqueductal and midbrain encephalitis has resulted in distortion and shrinkage of the infected tissue. The rostral portion of the aqueduct of Sylvius is dilated and leads to a blind pouch with complete occlusion just rostral to the fourth ventricle. Adjacent to the aqueduct the shrunken tissue has a whitish, granular appearance as a result of dystrophic calcification and gliosis.
mononuclear reaction was seen, the ependymal findings were marked. Much of the ependymal surface was denuded and replaced by mononuclear inflammatory cells and fibrous astrocytosis. Marked thinning of the cerebral cortex with lymphocyte-cuffed vessels and compression of the subcortical white matter were prominent (fig. 4). In the occipito-parietal cortex, there was only a very thin layer of neuronal cells and a gliotic molecular layer. In the sections through the aqueduct of Sylvius and rostral fourth ventricle, severe perivascular cuffing with lymphocytes and distended macrophages were prominent (fig. 5). Gliosis was observed in the quadrigeminal plate and sporadically throughout the tectal and pre-tectal areas in more caudal portions of the brainstem. Dystrophic calcification adjacent to the damaged ependymal lining was present in the periaqueductal region. Focal ependymal denudation, gliosis, periventricular inflammation, and patchy necrosis occurred throughout the brainstem adjacent to the fourth ventricle. The cerebellum showed only pre-myelination gliosis without evidence of encephalitis.
In summary, these findings indicate severe encephalitis affecting all portions of the brain except cerebellum with widespread ependymal damage, periependymal gliosis and aqueductal stenosis occurring in the midbrain region. Hydrocephalus as a result of this encephalitic process appears to be a t least partially attributable to the aqueductal stenosis. DISCUSSION
Influenza virus This report confirms reports of Fuccillo and Sever (‘73),Johnson and Johnson (‘69)and London et al. (‘75) that infections with influenza virus are established in fetal animals following experimental exposure. Virus was not recovered from organs other than those in contact with infected amniotic fluid. Ruben et al. (‘75) indicate that the virus has been isolated from lungs of abortuses of women with severe influenza infections. The portal of entry in post-natal human natural infections is known to be via cells of the respiratory tract. Therefore, this result is in concurrence. Because the virus has not spread to other organ tissues, these results are believed
62
MORELAND, GASKIN, SCHIMPFF, WOODARD AND OLSON
Fig. 4 The thinned cerebral cortical mantle is composed of compressed gray and white matter with perivascular lymphocytic cuffing, gliotic fibrous astrocytes and an inner denuded ependymal surface. Along the ventricular surface, inflammatory and glial cells form a multi-layered periventricular lining. x 100.
Fig. 5 The aqueduct of Sylvius and rostral fourth ventricle have the most severe inflammatory changes. Perivascular lymphocytic cuffing, distended macrophages, dystrophic calcification and intense gliosis are shown in this section immediately adjacent to the occluded aqueduct. x 100.
MONKEY FETAL MALFORMATIONS FROM MUMPS, WEE AND “FLU’
63
erally considered evidence that a viral agent has been present and an immunologic reaction has been evoked. Since antibody was not uniformly present in all fetuses from which virus was isolated we conclude that the elapsed time between infection and sample time was insufficient (7 days only) for detectable antibody production or that some fetuses were incapable of producing antibody at this stage of development (90-97 days gestation). The very low titers noted in most of the fetuses probably indicate that not enough time has elapsed Mumps virus for antibody production. Results confirm experimental fetal infecFA studies for detection of viral antigens in tions in hamsters as reported by Kilham and tissues were planned but as of this writing Margolis (‘75) and in rhesus monkeys as re- technical problems have prevented a conported by St. Geme and Van Pelt (‘74) and clusive determination on the significance of London et al. (’71). Further, the evidence pre- these studies. sented herein that in the primate fetus In attempting to limit the teratology experimumps virus invades essentially all organ tis- ments to the intracerebral inoculations of sues is a significant finding. This is true par- three animals with each virus (because of ticularly since it more closely mimics mumps scarcity, humane considerations and high cost infection in the human and since Johnson of nonhuman primates) it was realized that (‘68b) has specifically shown that in the ham- the sample size was marginal. Nevertheless, ster the virus does not seem to replicate in the data clearly point to a lack of teratogenic non-neural tissue. This assumes even greater effect of influenza virus while dramatically importance in light of St. Geme and Van Pelt’s showing fetal mortality and teratogenesis re(‘72) demonstration that virus could be re- sulting from mumps and WEE virus. covered from triturated fetal tissues of rhesus Mumps infection resulted in death of two of monkeys following intravenous inoculation of three fetuses. This number is insufficient to the dam, thus confirming transplacental in- draw firm conclusions and simply suggests fection. This suggests that the primate should additional studies to elucidate teratogenesis be more intensively studied as an animal and pathogenesis while supporting previous model which will simulate mumps infection a t observations that this virus results in a high incidence of fetal mortality. critical phases of development. The teratology study supports the observaThe 100%incidence of hydrocephalus in the tions of London et al. (‘71) that a high rate of three fetuses receiving WEE virus is striking, fetal mortality occurs in experimental infec- however, and strongly suggests the signifition in rhesus monkeys and of Siege1 et al. cant teratogenic potential of this virus. Although resulting in aqueductal stenosis, (‘66) that fetal mortality is increased in the pathology demonstrated in these fetal prihumans following natural mumps infection. mate specimens infected with WEE should not W E E virus be confused with noninflammatory aqueducThis is the first report of an experimental tal stenosis first reported by Johnson (‘68a) as animal study relating to fetal infections and a results of mumps inoculation in suckling clearly shows that (1) WEE virus infection hamsters. In those experiments, infection has can be established in fetal primates and (2) been shown to be almost exclusively limited to infection of fetuses with WEE virus may pro- ependymal cells and choroid plexus. Subseduce a severe encephalitis resulting in hydro- quent loss of damaged ependymal cells into cephalus. Considering the fact that infection the aqueduct and the associated reaction rewith this virus in adult humans is often sub- sults in obstructive hydrocephalus. No spread clinical these findings become increasingly of the mumps virus to the brain parenchyma significant and t h e implications assume was demonstrated nor were histologic findings suggestive of generalized encephalitis. The reserious proportions. sults described here demonstrate widespread General inflammatory histologic changes throughout The presence of antibodies in serum is gen- the central nervous system parenchymal tis-
to support the indications that fetal infection with influenza viruses does not lead to development of congenital malformations. This latter conclusion was further supported by results of the teratology experiment in which no malformations nor fetal deaths occurred following intracerebral instillation into the fetal brain. These results failed to confirm the observations of London et al. (’75) that intracerebral inoculation of influenza A virus caused hydrocephalus.
64
MORELAND, GASKIN, SCHIMPFF, WOODARD AND OLSON
sue indicative of severe encephalitis. In addition, ependymal denudation, periventricular inflammation and subsequent aqueductal stenosis resulted in severe obstructive hydrocephalus. It is not known if alterations in dosage, rout of inoculation, age of fetus a t exposure, or previous maternal immunity might restrict WEE infection to ependymal cells, thereby resulting in obstructive aqueductal stenosis not accompanied by generalized encephalitis. Based on the growing body of information which strongly suggests that obstructive hydrocephalus may be the result of early fetal ependymal infection, specific high risk infections need to be identified and methods devised to reduce fetal morbidity. This identification of WEE virus as a significant teratogenic agent has added to the growing number of experimentally proven teratogenic viral infections which require further evaluation; i t is particularly important because of reports of transplacental WEE human fetal infections. ACKNOWLEDGMENTS
The authors wish to acknowledge the advice and direction given by Doctor William T. London of the National Institute of Neurological, Communicative Disorders and Stroke who served as the Project officer for the contract. Also, we acknowledge the invaluable technical assistance of Marie Powell and Glenda Hall. LITERATURE CITED Cohen, R., R. E. O'Connor, T. E. Townsend, P. A. Webb and R. W. McKey 1953 Western equine encephalomyelitis: Clinical observations in infants and children. J. Pediatrics, 43: 26-34. Coops, S. C., and L. E. Giddings 1959 Transplacental transmission of Western equine encephalitis. Report of a case. Pediatrics, 24: 31-33. Ennis, F. A., H. E. Hopps, R. D. Douglas and H. M. Meyer 1969 Hydrocephalus in hamsters: Induction by natural attenuated mumps virus. J. Infect. Dis., 119: 75-79. Fuccillo, D. A., and J. L.Sever 1973 Viral teratology. Bacteriol. Rev., 37: 19-31. Hamburger, V., and K.Habel 1947 Teratogenic and lethal
effects of influenza A and mumps viruses on early chick embryos. Proc. SOC. Exp. Biol., 66: 608-617. Hammon, W. M., and G.E. Sather 1969 Arboviruses. In: Diagnostic Procedures for Viral and Rickettsial Infections. E. H. Lennette and N. J. Schmidt, eds. American Public Health Association, Inc., New York, pp. 227-280. Henle, W. 1969 Mumps virus. In: Diagnostic Procedures for Viral and Rickettsial Infections. E. H. Lennette and N. J . Schmidt, eds. American Public Health Association, Inc., New York, pp. 457-482. Johnson, R. E., and K. P.Johnson 1969 Hydrocephalus as a sequella of experimental myxovirus infections. Exp. Molecular Path., 20: 68. Johnson, R. T. 1968a Hydrocephalus following viral infection: The pathology of aqueductal stenosis developing after experimental mumps virus infection. J. Neuropathol. Exp. Neurol., 27: 591-606. 1968b Mumps virus encephalitis in the hamster: Studies of the inflammatory response and noncytopathic infection of neurons. J. Neuropath. and Experiment. Neurol., 27: 80-95. Kilham, L., and G. Margolis 1975 Induction of congenital hydrocephalus in hamsters with attenuated and natural strains of mumps virus. J. Infect. Dis., 132: 462-466. London, W. T., B. Curfman and J. L. Sever 1971 Mumps infection of t h e rhesus monkey fetus. Teratology, 4: 234 (Abstract). London, W. T., D. A. Fuccillo, J. L. Sever and S. G. Kent 1975 Influenza virus as a teratogen in rhesus monkeys. Nature, 255: 483-484. MacKenzie, J. S.,and M. Houghton 1974 Influenza infections during pregnancy: Association with congenital malformations and with subsequent neoplasms in children, and potential hazards of live virus vaccines. Bacteriol. Rev., 38: 356-370. Medovy, H. 1943 Western equine encephalomyelitis in infants. J. Pediatrics, 22: 308-318. Robinson, R. O.,and W. R. Dowdle 1969 Influenza viruses. In: Diagnostic Procedures for Viral and Rickettsial Infections. E. H. Lennette and N. J. Schmidt, eds. American Public Health Association Inc., New York, pp. 414-433. Ruben, F. L.,A. Winkelstein and R. E. Sabbagha 1975 In utero sensitization with influenza virus in man. Proc. Soc. Exp. Biol.Med., 149: 881-883. Shinefield, H. R., and T. E. Townsend 1953 Transplacental transmission of western equine encephalomyelitis. J. Pediatrics, 43; 21-25. Siegel, M., H. T. Fuerst and N. S. Peress 1966 Comparative fetal mortality in maternal virus diseases. N. Eng. J. Med., 274: 768-771. Spertzel, R. O.,C. L. Crabbs and R. E. Vaughn 1972 Transplacental transmission of Venezuelan equine e n cephalomyelitis virus in mice. Infect. Immun., 6: 339-343. St. Geme, J. W. Jr., and L. F. Van Pelt 1974 Fetal and postnatal growth retardation association with gestational mumps virus infection of the rhesus monkey. Lab. Animal Sci., 24: 895-899.
