720
Induction of Encephalitis in SJL Mice by Intranasal Infection with Herpes Simplex Virus Type 1: A Possible Model of Herpes Simplex Encephalitis in Humans Stephen J. Hudson, Richard D. Dix, and J. Wayne Streilein
Departments of Microbiology and Immunology, Ophthalmology, and Neurology, University of Miami School of Medicine, Miami, Florida
Herpes simplex encephalitis (HSE) is an acute, rapidly progressive encephalitis that almost invariably ends in death. In humans, the disease occurs typically in individuals who are putatively immunocompetent, that is, with no apparent immune deficiency. In fact, the central nervous system (CNS) infections with herpes simplex virus type 1 (HSV-l) that occur in immunocompromised individuals frequently display a different clinical picture, characterized by a slowly evolving, subacute course [1-4]. Histopathologic analyses of brains of immunocompetent patients dying of HSE show highly distinctive lesions comprised of hemorrhage and necrosis that are focal in nature and localized to the inferior frontal-temporal lobe and to the limbic system [5-7]; other areas of the brain are relatively spared [8, 9]. By contrast, microscopic studies of HSV-I-infected brains from immunocompromised individuals with encephalitis fail to show any focal or localizing signs; instead, mild to moderate histopathologic changes are dispersed diffusely throughout the brain [2, 4]. The strikingly different patterns of clinical disease and histologic findings that characterize HSV-I infections of the brain in immunologically normal, as opposed to immunologically compromised, individuals led Price et al. [4] to suggest that the formation of typical focal lesions in the temporal lobes of
Received 6 August 1990; revised 8 November 1990. Financial support: National Institutes of Health (EY-05678, EY-06662, I-PO-NS25569-01, and EY-07021-13 [to SJ.H.]), and the Joe and Emily Lowe Foundation, Palm Beach, FL. Reprints or correspondence: Dr. Stephen 1. Hudson, P.o. Box 016960 (R138), Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33101. The Journal of Infectious Diseases 1991;163:720-,727 © 1991 by The University of Chicago. All rights reserved. 0022-1899/91/6304-0007$01.00
brains of patients dying of typical HSE is dependent upon the presence of an intact immune system and response. It has not been possible to test this provocative proposal because of the lack of an immunologically defined animal model that faithfully mimics the human disease [l0-l12]. We initiated experiments designed to produce in laboratory mice a form of HSV-l-induced encephalitis that is similar, if not identical, to human HSE. This approach led us to test whether intranasal instillation of an HSV-l clinical isolated obtained from a patient with HSE could cause focal disease in the brains of a diverse array of genetically defined strains of mice. The strains were selected for various reasons, including previously documented susceptibility or resistance to systemic infection with HSV-I [13, 14], susceptibility to CNS disease caused by immunopathogenic responses to other viruses [IS-17], susceptibility to autoimmune CNS disease [18, 19]. The intranasal route of inoculation was chosen because it is thought to be an important route of infection in human HSE [6, 20].
Materials and Methods Animalsand viralinoculations. Nine strains of inbred mice (A/J, AKR, A.SW, BALB/c, BlO.S, C3H/HeJ, C57BLl6, DBA/2, SJLlJ) were obtained from the Jackson Laboratories (Bar Harbor, ME). In addition, C.B17-SCID mice were supplied by 1. Stein-Streilein (Department of Medicine, University of Miami School of Medicine). Female mice were used for all studies. All animals were 8-10 weeks old except for SJL strain mice (10-12 weeks old) and C.BI7-SCID mice (12-16 weeks old). Mice were anesthetized with pentobarbitol and a 2-J.L1 droplet of virus was dispensed into each nare. Doses of virus are listed in table 1. Viral dilutions were made in minimal essential media (MEM) , and the samples of viral inocula were titrated as described below to confirm the viral dose. Virusand cell culture. A well-described [21] clinical isolate of HSV-l (strain H129) was used in these studies. A viral stock was
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on June 9, 2015
Herpes simplex encephalitis (HSE) is characterized by focal lesions of hemorrhage and necrosis, primarily in the inferior temporal lobe. Since immunosuppressed patients with HSE lack the focal inflammatory changes and temporal lobe localization, it has been suggested that the immune system participates in the pathogenesis of HSE. Evaluation of this hypothesis has been impeded by the lack of an immunologically defined animal model that resembles the human disease. Toward this end, 10 strains of inbred mice were infected intranasally with a neurovirulent clinical isolate of herpes simplex virus type 1. Most mice died without localizing signs of disease in the central nervous system. However, a significant number of SJL mice had a pattern of encephalitis highly reminiscent of that described in humans. To our knowledge, this is the first murine model that faithfully mimics this human disease, and thus it affords the opportunity to study the immunopathogenesis of HSE.
1ID 1991;163 (April)
A Murine Model of Herpes Simplex Encephalitis
721
Table 1. Dose responses of various mouse strains to intranasal infection with herpes simplex virus type 1 (H 129).
Strain
n
SIL A/I
DBAI2
C3H/Hel
AKR
BALB/c
27
C57BL/6 BlO.S
* Mice
35 20
5-10 days
11-13 days
surviving'[
6 5 6 5 6 5 4 6 5 4 4 6 5 6 5 4 6 5 '4 6 6
59 5 100 50 94 100 47 95 100 65 65 100 14 100 93 44 98 100 52 26 25
19 0 0 25 1 0 0 0 0 9 35 0 0 0 0 0 0 0 0 0 0
22 95 0 25 3 0 53 5 0 26 0 0 86 0 7 56 2 0 48 74 75
% mice
sacrificed when moribund. Days = days after inoculation.
t Includes mice that recovered from clinical illness or never displayed clinical signs of disease.
prepared by infecting human embryonic lung (HEL) fibroblasts (cell line MRC-5) with virus at an input multiplicity of 0.01 pfu/cell. The infectivity of the stock was assayed on cultures of African green monkey kidney (Vero) cells (Flow Laboratories, Inglewood, CA) by the plaque assay method using a 2 % methyl cellulose overlay [22]. Results were obtained with virus from a single stock preparation with a titer of 2.25 x 108 pfu/rnl. Clinical and pathologic evaluation. All animals were examined daily for ruffled coats, labored breathing, ataxia, and seizures. Moribund animals were killed and their brains were examined for gross pathologic changes. Brains were sectioned along the midline and fixed in 10 % phosphate-buffered formalin for microscopic examination. Paraffin-embedded brain halves were evenly divided coronally into five blocks, and five coronal sections (6 pm each) were cut from each block. Tissue sections were stained with hematoxylin-eosin (HE) and histologic disease was assessed by determining the presence and distribution of focal inflammatory lesions of hemorrhage and necrosis in the brain. Selected mice were killed by perfusion with saline to determine the distribution of hemorrhage within the brain. Histochemistry. Brains were removed from moribund mice (6-8 animals per strain) and placed in a.C.T. compound (Miles, Elkhart, IN) before freezing. Six serial cryostat sections (6 /Lm each) were cut at 1.5-mm intervals throughout the brain and fixed in acetone (4°C) for 10 s. Rabbit anti-HSV-l and normal rabbit serum (Dako, Santa Barbara, CA) were both absorbed with mouse liver powder (Sigma, St. Louis) and diluted 1:140 before use. The binding of antiHSV-l serum to frozen tissue sectionswas detected by immunoperoxidase staining using the chromogen 3-amino-9-ethylcarbazole. Biotinylated goat anti-rabbit and avidin-conjugated horseradish per-
oxidase were used in accordance with the manufacturer's instruction (Biogenex, San Ramon, CA). Quantitation of infectious virus from CNS tissue. Brain halves were individually homogenized in MEM to yield 10% (wt/vol) homogenates. Plaque assays ofhomogenates (serially diluted) were done in duplicate and virus titers were expressed as plaque-forming units per gram (fresh weight) of tissue. Statistics. Regression lines corresponding to survival time ofvarious HSV-l-infected mice were compared using the t test [23].
Results Clinical patterns of disease in genetically disparate mice infected intranasally with H~l. In the first set of experiments, a viral dose of I (1i pfu of HSV-I was administered to mice of each strain (excluding C.BI7-SCID). Animals from each group were evaluated for clinical and histopathologic evidence of encephalitis. Additional panels of mice of some strains were inoculated with 104 or 105 pfu of HSV-I when it was determined that 106 pfu resulted in 100% mortality before onset of signs of encephalitis. Regardless of mouse strain, the earliest signs of systemic disease were evident 5-7 days after inoculation when affectedanimals had ruffled coats, labored breathing, and hunched posture. The greatest frequency of imminent death (necessitating sacrifice) occurred 5-10 days after inoculation. In strains DBA/2, C3H/He1, AKR, A.SW, BALB/c, C57BL/6, and BIO.S, most animals that would ultimately die of viral infection died during this
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on June 9, 2015
C.BI7-SCID A.SW
54 22 17 12 34 3 15 20 20 23 20 6 7 6 27 9 50 3
% mice moribund *
Virus dose (loglO pfu)
722
Hudson et aI.
Figure 1. Hemorrhagic necrosisof the temporal area (arrow) 11 days after intranasalinoculation of SJL mouse with herpes simplex virus type 1. Bar = 2 mm.
regions of the cerebrum, brain stem, or cerebellum. Tissue sections prepared from surviving mice did not show FERNE lesions . Immunoperoxidase staining for viral antigens in the brains of SJL mice 7 days after inoculation (before the onset of FERNE) revealed that the localization of FERNE·lesions corresponded with the sites of active viral replication (figure 3). The brains of A/J mice that became moribund 11-13 days after inoculation also had eosinophilic necrosis of neurons within the pyriform and entorhinal cortices of the temporal area but lacked the intense inflammatory infiltrate and hemorrhage present in SJL mice . The brains of mice that died 5-10 days after inoculation did not show FERNE. For example, in BALB/c mice (figure 2b) necrosis of individual neural and glial cells was observed scattered throughout cerebrum and brain stem; the cerebellum was unremarkable. Inflammation was modest and diffusely apparent within the temporal area , and a mild cellular infiltrate was present in the adjacent layer of the meninges. However, localizing signs of focal involvement of the brain were not seen.
Quantitationofinfectious virus in the brains ofmoribund miceafterintranasal infection withHSV-J. The striking differences in the histopathology of SJL and other mouse strains led us to determine if there was a difference in the titer of infectious virus in the brains of these animals. Whole brains removed from moribund A/J, DBA/2 , C3H/HeJ, AKR, BALB/c, C57BLl6 (5-10 days after inoculation), or SJL mice (11-13 days after inoculation) were used to quantitate the amount of infectious virus by standard plaque assay technique [22]. Viral titers in whole brain were similar for SJL, C3H/HeJ, AKR, BALB/c, DBA, and C57BLl6 mice (table 2) even though SJL mice became moribund later than the other strains examined. However, SJL mice that became moribund early (5-10 days after inoculation) also had similar viral titers (data not shown) . Viral titrations of brain tissues from mice
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on June 9, 2015
interval (table 1). Many of these animals did not have clinical signs associated with encephalitis, such as ataxia and seizures. By contrast, a significant number of mice of strains A/J and SJL survived a few days longer and did not become moribund until 11-13 days after inoculation . Virtually all of these mice had clinical evidence of encephalitis manifested in the form of ataxia or seizures. While most of these animals died by day 12, a few survived to day 13. Animals that survived beyond day 13 did not die. Survivors included animals that had ocular disease and cutaneous lesions without evidence of encephalitis and those clinically normal throughout the observation period. SJL strain mice have a maturational defect in their immune system that prevents the expression of adult levels of delayedtype hypersensitivity until 10 weeks of age [24]. To determine if the difference in age between SJL mice (10-12 weeks) and the other strains of mice used (8-10 weeks) could account for the patterns of pathology observed, panels of 1O-12-weekold mice (A/J, BALB/c, C57BLl6) were infected with HSV-l and the clinical and histologic pattern of disease was assessed. In these experiments the pattern of pathology observed correlated strictly with mouse strain and was not affected by age difference. Gross pathology. Brains from moribund mice and survivors were examined for gross and microscopic pathologic changes. Gross pathologic changes were not observed in animals that became acutely ill and were sacrificed before day 10 after inoculation. By contrast, SJL mice with clinical signs of encephalitis that became moribund 10-12 days after inoculation showed bilateral softening of the temporal area. Many of these animals also displayed hemorrhagic necrosis of the temporal areas that was usually bilateral, but the intensity of hemorrhage varied considerably from side to side. Perfusion of selected SJL mice showed that hemorrhage, when present, was localized exclusively to the temporal area of the brain (figure 1). There was no evidence of hemorrhage at any other site . Microscopic pathology. HE-stained brain sections from SJL mice had a histopathologic pattern similar to that described in brains of humans with HSE [5-7]. Focal necrotic and inflammatory lesions were seen only within the hippocampus and the entorhinal and pyriform cortexes of the temporal area (figure 2a). In these sites alone , eosinophilic necrosis of neurons, extensive hemorrhage, and intense infiltration with inflammatory cells were observed. The cellular infiltrate comprised largely monocytes and lymphocytes, which formed prominent perivascular cuffs with the lesion. We have designated this histologic pattern of disease "focal entorhinal necrotizing encephalitis" (FERNE) to distinguish it from histologically different forms of encephalitis seen in the brains of other mouse strains (described later) . In other regions of the cerebral cortex of SJL mice, diffuse hypercellularity and "activated" microglial cells were also seen. Importantly, no focal necrotic lesions were observed in other
JID 1991;163 (April)
JID 1991;163 (April)
A Murine Model of Herpes Simplex Encephalitis
723
250 ILm.
.- . .'
with moderate disease yielded only low titers of virus; survivors had none. Two of the five A/J mice assayed showed "-'1O-fold higher amounts of virus than did the other strains assayed. We failed to find an association between the quantity of infectious virus present in the brains of SJL mice, which developed FERNE, and the amount of virus in brains of other mouse strains that also became moribund but had no focal lesions.
tranasal infection with HSV-l were stained for viral antigens by the immunoperoxidase technique. Areas that stained positive for replicating virus were localized within projections of the olfactory pathway. Numerous infected cells were detected in the entorhinal and pyriform cortexes in both SJL (figure 4a) and BALB/c mice (figure 4b) .
Localization ofviral antigens in the brain after intranasal infectionwith HSV-l. Because differences in the quantity of
of the immune system in the pathogenesis of HSE, we also infected C.BI7-SCID mice intranasally with H}' pfu of HSV-l. This strain has a mutation that prevents T and B lymphocytes from generating receptors for antigen-either antibodies or T cell surface receptors. As a consequence, C.B17-SCID mice are deficient in adaptive immunity [25, 26]. All C.BI7-SCID mice infected intranasally with HSV-l 00-
virus could not account for the different histopathologic patterns among SJL and other mouse strains (e.g., BALB/c), we next sought to determine if there was a difference in the distribution of viral antigens in the brains of these animals. Coronal sections from SJL and BALB/c mice 7 days after in-
Patterns ofdisease in C Bl7-SCID mice infected intranasally with HSV-l. Because of interest in the possible involvement
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on June 9, 2015
Figure 2. Coronal sections (hematoxylin-eosin stain) from temporal area of brains from SJL mice with acute disease after intranasal inoculation with herpes simplex virus type 1. A, Focal necrosis of pyriform cortex (enclosed area , arrow) 11 days after infection. In contrast, 7 days after infection BALB/c mice (B) show mild inflammation and meningitis but no focal lesions. Bar =
Hudson et aI.
724
JID 1991;163 (April)
"
...
:
A
1 (HSV-l) antigens in olfactorycenterswithintemporal area of brain from SJL mouse7 daysafter intranasalinoculation with HSV-l. Bar = 500 !-lm .
.-
Table 2. Number of mice with positiveviral culture after intranasalinoculation withherpes simplex virustype 1 (HSV-I) (H129).
B
.No . of mice with HSV-I titers of
IOLI04 104-10 5 105-106 106-107 lOLI08 Strain
n
AKR DBAI2 SJL BALB/c C3H /HeJ C57BLl6 A /J C .B17-SClD
7 5 5 5 6 3 5 5
(p ful g fresh wt)
0 0 1 0 0 0 0 0
2
0 0 0 1
0 0 0
3 4 2 3 .2
2 I 2
2 3 2
2 3
0 0 0 0 0 0 2 1
came infected with this virus. Some animals became moribund as early as 6 days after inoculation; however, a large fraction of animals did not become severely ill and moribund until as late as 11-13 days after inoculation (table 1). When moribund mice from this latter group were sacrificed, the distribution of histopathologic changes observed in their brains was strikingly similar to those observed in BALB/c mouse brains and consisted chiefly of diffuse and scattered necrosis of individual neurons throughout the cerebrum and brain stem (data not shown) . However, the intensity of cellular necrosis rivaled that seen in some A/J mice, which had similar virus titers in the CNS (table 2). Importantly, there were no focal CNS lesions and no evidence oflocalized pathologic changes in any region of their brains. It is unlikely that the extended survival times of C.BI7-SCID mice was due to differences in
Figure 4. Light micrographs of numerous herpes simplex virus type 1 (HSV-l)-infected cel1s (arrows, representative cells)in pyriformarea of brains from SJL (A)and BALBfc (B) mice 7 daysafter intranasal inoculation with HSV-l. Bar = 100 !-lm.
genetic background alone, since a high frequency of these animals survived longer than any of the immunocompetent strains evaluated. Association ofsurvival time with the clinical pattern ofdisease and FERNE. The data obtained from mice infected with viral doses that resulted in the death of at least some of the infected animals (table 1) were used to construct a survival curve (figure 5). This analysis showed two significantly different (P < .002) rates of mortality among the mouse strains infected with HSV-l. The slope of the curve depicting mice that died between 5 and 9 days is most steep (r = 0.99); the slope that described the death rate between days 10 and 13 is less steep (r = 0.93) and the difference between the two slopes is highly significant (P < .002) . Survival times were highly mouse-strain dependent, with SJL and A/J mice representing the largest fraction of animals that died 10-13 days after inoculation (table I). Interestingly, these mice presented the most consistent clinical manifestations of encephalitis, and FERNE was found exclusively in SJL mice that became moribund during this interval. These findings imply that animals infected intranasally with HSV-I develop two
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on June 9, 2015
Figure 3. Immunoperoxidase staining forherpessimplex virus type
A Murine Model of Herpes Simplex Encephalitis
JID 1991;163 (April)
350 ,--u==~:-:------r========;iT'" 20
300 15 01
c:
:~
250
w
~
Z
a:
:J
en
w 10 u,
CD
0
:?: 0
z
0
z
200
5 150
2
--4..............-...._.--tlI--.-----r------'l...-......-
4
6
8
10
12
0
14
Day post-infection
Figure 5. Survival and frequency of encephalitis in various inbred mouse strains after intranasal inoculation with herpes simplex virus type 1 (HSV-l). Mice were infected at virus doses that resulted in survival rates of 3 %-95 %. FERNE = focal entorhinal necrotizing encephalitis.
temporally distinct, and perhaps pathogenically unique, types of disease, only one of which is a possible model of HSE. As anticipated, mortality rates were the highest for C.B17SCID mice. Because of their immunodeficient status, the data from these mice are not included in the survival curve. It is particularly important, however, to examine the actual pattern of survival among these grossly immune incompetent mice. Note that in table 1, 7 of20'C.BI7-SCID mice survived the 5-10-day interval only to die 11-13 days after inoculation. This indicates that immunoincompetent mice can survive a lethal infection with intranasal HSV-l longer than some immunocompetent mice (BALB/c, DBAI2) infected with a viral dose of equal magnitude. Discussion By surveying many genetically disparate inbred strains of mice, we have identified the SJL mouse as a potential model of human HSE. The characteristic features of human HSE that we feel must be reproduced in any suitable animal model include (1) acute onset, (2) usually fatal outcome, (3) localization of pathologic CNS changes preferentially to the inferior temporal area and limbic system, (4) focal inflammatory lesions of necrosis and hemorrhage with this tissue distribution, and (5) occurrence in an apparently immunocompetent host. The disease produced by intranasal infection of SJL with HSV-1 (which we described here) fulfills most of these criteria. A high proportion ofSJL mice died 11-13 days after inocula-
tion after suffering obvious clinical signs of CNS involvement. When their brains were examined microscopically, focal necrotic and hemorrhagic lesions were found, located exclusively within the inferior temporal area. Since the recipients were not exposed to immunosuppressive agents before intranasal infection, and since they were adults (10-12 weeks old), they were fully immunocompetent when virus was instilled. Thus, the criterion of occurrence in an otherwise immunologically normal individual was met. We anticipate that future study of this animal model will shed new light on the pathogenesis of human HSE. Experimental induction of HSV-1 infections of the brains of mice and other laboratory animals has been done many times previously. However, none of the previous experimental strategies produced clinical and histopathologic findings of the type reported here using both an immunologically described model and a proposed route of infection for the human disease [6, 10]. Stroop and Schaefer [27] and Schlitt et al. [28] have both produced focal lesions restricted to the temporal lobes using rabbit models of HSE. However, the use of immunosuppressive agents [27] or the paucity of rabbit immunologic reagents [27, 28] make these models impractical for studying the putative role of the immune response in HSE. Direct intracerebral inoculation of HSV-1 usually causes a diffuse encephalitis that is lethal in 7-10 days [21, 29]. However, McFarland et al. [11] have reported extended survival times and produced focal lesions in the limbic systems of mice after microinjection of a low-virulence strain of HSV-l directly into the hippocampus. Damage to the blood-brain barrier and trauma to surrounding CNS tissue by this artificial method of infection likely has an important impact on the ensuing immune response. Injection of HSV-1 subcutaneously and intraperitonea1ly causes encephalitis in some recipients, depending upon factors such as the number ofinfectious viral particles in the inoculum, the strain of virus, and the genetic constitution of the host [30]. Almost invariably, the pattern of CNS involvement that has been described is diffuse, without localizing or pathologic signs. The intranasal route of infection has also been used previously to cause encephalitis in mice [29, 31, 31a]. However, perhaps because of the viral dose used, or due to the genetic constitution of the recipient, encephalitis of the unique form that occurs in SJL mice, and that resembles human HSE, was not observed. Our findings that moribund SJL and BALB/c mice have comparable viral titers in the brain and the same localization of viral antigens to the primary olfactory areas emphasize the point that host genetic factors playa critical role in determining the pathologic consequence of infection in the brain. Depending upon the dose of virus, a variable proportion of mice of each strain became lethargic, hunched, and moribund 5-10 days after inoculation. Most of these animals had no clinical signs of encephalitis, nor did their brains display FERNE-type lesions. In addition to animals showing this type of disease early after intranasal infection, an equal propor-
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on June 9, 2015
100 -+-
725
726
Hudson et al.
HSV-l. Certain immunologic features of SJL, although their genetic basis may not be well understood, are potentially relevant to development of HSE. For example, SJL mice infected with Theiler's virus develop a demyelinating CNS disease in which an immunopathogenic process appears to intervene as the proximate cause of damage to myelinated nerve fibers [33, 34]. Moreover, among inbred mouse strains, SJL is especially susceptible to the development of experimental autoimmune encephalitis (EAE) induced by immunization with myelin basic protein [35, 36]. Although postulating a relationship between the pathogenesis ofHSE and that of Theiler's demyelination or EAE is premature at this time, the link between an immune response to a viral or tissue antigen and the inflammation that becomes targeted to the brain is similar for all. SJL mice are also of interest because of two other immunologic abnormalities they display. First, Stohlman et al. [24] documented that the ontogenetic appearance of the capacity to develop and express delayed hypersensitivity is delayed in SJL mice relative to other strains. Not until these animals are 10-12 weeks old can they respond to an antigenic challenge with delayed hypersensitivity that rivals the intensity of that observed in other mice. The maturational defect, although still imprecisely understood, appears to be expressed in macrophages [37]. Second, the genome of SJL mice contains a large deletion of genetic material from the locus that encodes the variable part of the {3 chain of the T cell receptor [38]. At this point, neither aspect of the immune system of SJL mice translates directly into an explanation for their susceptibility to HSE. However, since we anticipate that delayed hypersensitivity is likely to be a major effector modality in the expression ofHSE, both defects may be pertinent. As we attempt to understand the immunopathogenesis of HSE in SJL mice, these features will figure prominently in our experimental approach.
Acknowledgment We thank M. Norenberg for assistance in the histologic analysis and Maria Saenz for expert technical assistance.
References 1. Dix RD, Bredesin DE. Vial infections in acquired immunodeficiency syndrome. In: Rosenblum ML, Levy RM, Bredesen DE, eds. AIDS and the nervous system. New York: Raven Press, 1988:221-61. 2. Hotson JR, Pedley TA. Neurological complications of cardiac transplantation. Brain 1976;99:673-94. 3. Pepose JS, HBOOrne LH, Cancilla PA, Foo RY. Concurrent herpes simplex and cytomegalovirus retinitis and encephalitis in the acquired immune deficiency syndrome (AIDS). Ophthalmology 1984;91: 1669-77. 4. Price P, Chernik NL, Horta-Barbosa L, Posner JB. Herpes encephalitis in an anergic patient. Am J Med 1973;54:222-7. 5. Clizer EE, Loannides G. Herpes simplex encephalitis. AJR 1971; 112:273-5. 6. Esiri MM. Herpes simplex encephalitis-an immunohistological study
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on June 9, 2015
tion of mice of some strains (SJL and A/J) became moribund later, 11-13 days after inoculation. Many of these animals developed clear-cut clinical evidence of encephalitis and, when sacrificed, the brains of SJL mice showed the changes typical of FERNE. The brains of A/J mice showed changes in the temporal area characterized by individual cell necrosis, but the intense inflammatory component of FERNE was absent. The presence of high viral titers in some A/J mice suggests that this form of encephalitis may be mediated by direct viral cytopathology, with less of a contribution by the immune response. A third group of animals fits into neither of these two categories; these animals developed either no clinical disease or transient clinical symptoms (usually without CNS signs) . from which they recovered. Histologic evaluation of their brains was unremarkable. These three distinct outcomes after intranasal infection of mice with HSV-1 occurred even though all animals used for these experiments were considered immunocompetent. The importance of this observation is underscored by the outcome of intranasal infection of C.B17-SCID mice in whom adaptive immunity [25, 26] cannot take place because of a genetic defect that precludes their lymphocytes from generating antibodies or lymphocyte surface receptors for antigen. All C.B17-SCID mice infected intranasally with HSV-1 became infected with this virus, becoming moribund as early as day 6 or as late as days 11-13 after inoculation. The death of these latter mice corresponds with the time during which SJL mice developed FERNE. However, the histopathologic changes observed in the brains ofC.B17-SCID mice resembled those observed in brains of BALB/c, not SJL, mice. There were no focal lesions or localized pathologic changes in any region of their brains. This result implicates an immunopathogenic process in the development of FERNE and supports the original hypothesis of Price et al. [4] concerning the participation of the immune system in HSE. We presume that the diffuse pattern of pathologic changes observed in the brains of C.B17SCID mice, without focal necrosis, reflects direct cytopathic effectsof virus in the absence of participation by immune effectors. In addition, while a significant number of C.B17-SCID mice survived beyond day 10, a significant number of "normal" BALB/c mice died 5-10 days after intranasal infection. Although we do not yet know the cause of death in these BALB/c mice, the fact that they died earlier than many similarly infected C.B17-SCID mice implies that the early death of BALB/c mice may also be mediated in part by an immunopathogenic process. Now that HSE can be induced in experimental form in SJL mice, we are interested in understanding the nature of the genetic factors that operate in these mice to render HSE possible. It is known that the major histocompatability complex does not seem to influence susceptibility to HSV infections in mice [32]. That appears true as wellfor HSE, since in our experiment SJL, but not the H-2 congenic B10.S or A.SW strains of mice, developed HSE after intranasal infection with
JID 1991;163 (April)
JID 1991;163 (April)
7.
8. 9.
10. 11.
13.
14. 15.
16.
17.
18.
19.
20. 21.
22. 23.
of the distribution of viral antigens within the brain. J Neurol Sci 1982;4:209-26. Nahmias Al, Whitley RJ, Visintine AN, Taki Y, Alford CA Jr, Collaborative Antiviral Study Group. Herpes simplex virus encephalitis: laboratory evaluations and their diagnostic significance. J Infect Dis 1982;145:829-36. Adams H, Miller D. Herpes simplex encephal itis - a clinical and pathological analysis of 22 cases. Postgrad Med J 1973;49:393-7. Haymaker W, Smith MG, Van Bogaert L, De Chenar C. Pathology of the viral disease in man characterized by nuclear inclusions. In: Field WS, Blatter RL, eds. Viral encephalitis. Springfield, IL: CC Thomas, 1958:95-104. Elizan TS, Maker H, Yahr MD. Neurotransmitter synthesizing enzymes in experimental viral encephalitis. J Neural Trans 1983;57:139-47. McFarland DJ, Sikora E, Hotchin J. The production of focal herpes encephalitis in mice by stereotaxic inoculation of virus. J Neurol Sci 1986;72:307-18. Sckizawa T, Openshaw H. Encephalitis resulting from reactivation of latent herpes simplex virus in mice. J Virol 1986;50:263-6. Kastrukoff LF, Lau AS, Puterman ML. Genetics of natural resistance to herpes simplex virus type 1 latent infection of the peripheral nervous system in mice. J Gen Virol 1986;67:613-21. Lopez C. Genetics of natural resistance to herpesvirus infection in mice. Nature 1975;258:152-3. Allen JE, Doherty Pc. Consequences of a single Ir gene defect for the pathogenesis of lymphocytic choriomeningitis. Immunogenetics 1985;21:581-90. Clatch RJ, Melvold RW, Dal Canto MC, Miller SD, Lipton HL. The Theiler's murine encephalomyelitis virus (TMEV) model for multiple sclerosis shows a strong influence of the murine equivalents of HLA-A, B, and ~. J Neuroimmunol 1987;15:121-35. Lipton HL, Dal Canto MC. Susceptibility of inbred mice to chronic central nervous system infection by Theiler's murine encephalomyelitis virus. Infect Immun 1979;26:369-74. Tabira T. Autoimmune demyelination in the central nervous system. In: Raine CS, ed. Advances in neuroimmunology. Vol. 540. New York: New York Academy of Sciences, 1988:187-201. Touhy VK, Sobel RA, Lees MB. Myelin proteolipid protein-induced experimental allergic encephalomyelitis - variation of disease expression in different strains of mice. '.r Immunol 1988;140:1868-73. Johnson RT. Viral infections of the central nervous system. New York: Raven Press, 1982:135-8. Dix RD, McKendall RR, Barenger RJ. Comparative neurovirulence of herpes simplex virus type 1 strains after peripheral or intracerebral inoculation of BALB/c mice. Infect Immun 1983;40:103-12. Dreesman GR, Benyesh-Melnick M. Spectrum of human cytomegalovirus compliment-fixing antigens. J Immunol 1967;99:1106-14. Glantz SA. Primer of biostatistics. New York: McGraw-Hill, 1981: 180-227.
727
24. Stohlman SA, Matsushima GK, Casteel N, Frelinger JA. The defect in delayed-type hypersensitivity of young adult SJL mice is due to a lack of functional antigen presenting cells. Eur J Immunol 1985;15:913-6. 25. Bosma GC, Custer RP, Bosma MJ. A severecombined immunodeficiency mutation in the mouse. Nature 1983;301:527-30. 26. Dorshkind K, Keller GM, Philips RA, et al. Functional status of cells from lymphoid and myeloid tissues in mice with severe combined immunodeficiency disease. J Immunol 1984;132:1804-8. 27. Stroop WG, Schaefer DC. Production of encephalitis restricted to the temporal lobes by experimental reactivation of herpes simplex virus. J Infect Dis 1986;153:721-31. 28. Schlitt M, Lakerman AD, Wilson ER, et al. A rabbit model of focal herpes simplex encephalitis. J Infect Dis 1986;153:732-5. 29. Anderson JR, Field HJ. The distribution of herpes simplex type 1 antigen in the central nervous system after different routes of inoculation. J Neurol Sci 1983;60:181-95. 30. Dix RD, Mills J. Experimental mouse models of herpes simplex virus infection. In Zak 0, Sande MA, eds. Experimental models in antimicrobial chemotherapy. Vol. 2. London: Academic Press, 1986: 219-57. 31. De Clercq E, Miroslav L. Intranasal challenge of mice with herpes simplex virus: an experimental model for evaluation of the efficacy of antiviral drugs. J Infect Dis 1976;133:A226-36. 3la. TomlinsonAH, Esiri MM. Herpes simplex encephalitis-immunohistological demonstration of spread of virus via olfactory pathways in mice. J Neurol Sci 1983;60:473-84. 32. Lopez C. Resistance to HSV-l in the mouse is governed by two major, independently segregating, non-H-2 loci. Immunogenetics 1980; 11:87-92. 33. Clatch RJ, Lipton HL, Miller SD. Characterization of Theiler's murine encephalomyelitis virus (TMEV)-specific delayed type hypersensitivity responses in TMEV-induced demyelinating disease: correlation with clinical signs. J Immunol 1986;136:920-27. 34. Clatch RJ, Melvold RW, Miller SD, Lipton HL. Theiler's murine encephalomyelitisvirus (TMEV)-induced demyelinatingdisease in mice is influenced by the H-2D region: correlation with TMEV-specific delayed type hypersensitivity. J Immunol 1985;135:1408-14. 35. Kennedy MK, Dal Canto MC, Trooter JL, Miller SD. Specific immune regulation of chronic-relapsing experimental allergic encephalitis in mice. J Immunol 1988;141:2986-93. 36. Pettinelli CB, Fritz RB, Jen Chou CH, McFarlin DE. Encephalitogenic activity of myelin basic protein in the SJL mouse. J Immunol 1982;129:1209-11. 37. Matsushima GK, Stohlman SA. Maturation of the delayed-type hypersensitivity response in SJL mice: absence of effector cell induction. Eur J Immunol 1988;18:1411-16. 38. Huppi KE, D'Hoostelaere LA, Mock BA, et al. T-cell receptor VT(3 genes in natural populations of mice. Immunogenetics 1988;27:51-6.
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on June 9, 2015
12.
A Murine Model of Herpes Simplex Encephalitis
Induction of Encephalitis in SJL Mice by Intranasal Infection with Herpes Simplex Virus Type 1: A Possible Model of Herpes Simplex Encephalitis in Humans Stephen J. Hudson, Richard D. Dix, and J. Wayne Streilein
Departments of Microbiology and Immunology, Ophthalmology, and Neurology, University of Miami School of Medicine, Miami, Florida
Herpes simplex encephalitis (HSE) is an acute, rapidly progressive encephalitis that almost invariably ends in death. In humans, the disease occurs typically in individuals who are putatively immunocompetent, that is, with no apparent immune deficiency. In fact, the central nervous system (CNS) infections with herpes simplex virus type 1 (HSV-l) that occur in immunocompromised individuals frequently display a different clinical picture, characterized by a slowly evolving, subacute course [1-4]. Histopathologic analyses of brains of immunocompetent patients dying of HSE show highly distinctive lesions comprised of hemorrhage and necrosis that are focal in nature and localized to the inferior frontal-temporal lobe and to the limbic system [5-7]; other areas of the brain are relatively spared [8, 9]. By contrast, microscopic studies of HSV-I-infected brains from immunocompromised individuals with encephalitis fail to show any focal or localizing signs; instead, mild to moderate histopathologic changes are dispersed diffusely throughout the brain [2, 4]. The strikingly different patterns of clinical disease and histologic findings that characterize HSV-I infections of the brain in immunologically normal, as opposed to immunologically compromised, individuals led Price et al. [4] to suggest that the formation of typical focal lesions in the temporal lobes of
Received 6 August 1990; revised 8 November 1990. Financial support: National Institutes of Health (EY-05678, EY-06662, I-PO-NS25569-01, and EY-07021-13 [to SJ.H.]), and the Joe and Emily Lowe Foundation, Palm Beach, FL. Reprints or correspondence: Dr. Stephen 1. Hudson, P.o. Box 016960 (R138), Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33101. The Journal of Infectious Diseases 1991;163:720-,727 © 1991 by The University of Chicago. All rights reserved. 0022-1899/91/6304-0007$01.00
brains of patients dying of typical HSE is dependent upon the presence of an intact immune system and response. It has not been possible to test this provocative proposal because of the lack of an immunologically defined animal model that faithfully mimics the human disease [l0-l12]. We initiated experiments designed to produce in laboratory mice a form of HSV-l-induced encephalitis that is similar, if not identical, to human HSE. This approach led us to test whether intranasal instillation of an HSV-l clinical isolated obtained from a patient with HSE could cause focal disease in the brains of a diverse array of genetically defined strains of mice. The strains were selected for various reasons, including previously documented susceptibility or resistance to systemic infection with HSV-I [13, 14], susceptibility to CNS disease caused by immunopathogenic responses to other viruses [IS-17], susceptibility to autoimmune CNS disease [18, 19]. The intranasal route of inoculation was chosen because it is thought to be an important route of infection in human HSE [6, 20].
Materials and Methods Animalsand viralinoculations. Nine strains of inbred mice (A/J, AKR, A.SW, BALB/c, BlO.S, C3H/HeJ, C57BLl6, DBA/2, SJLlJ) were obtained from the Jackson Laboratories (Bar Harbor, ME). In addition, C.B17-SCID mice were supplied by 1. Stein-Streilein (Department of Medicine, University of Miami School of Medicine). Female mice were used for all studies. All animals were 8-10 weeks old except for SJL strain mice (10-12 weeks old) and C.BI7-SCID mice (12-16 weeks old). Mice were anesthetized with pentobarbitol and a 2-J.L1 droplet of virus was dispensed into each nare. Doses of virus are listed in table 1. Viral dilutions were made in minimal essential media (MEM) , and the samples of viral inocula were titrated as described below to confirm the viral dose. Virusand cell culture. A well-described [21] clinical isolate of HSV-l (strain H129) was used in these studies. A viral stock was
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on June 9, 2015
Herpes simplex encephalitis (HSE) is characterized by focal lesions of hemorrhage and necrosis, primarily in the inferior temporal lobe. Since immunosuppressed patients with HSE lack the focal inflammatory changes and temporal lobe localization, it has been suggested that the immune system participates in the pathogenesis of HSE. Evaluation of this hypothesis has been impeded by the lack of an immunologically defined animal model that resembles the human disease. Toward this end, 10 strains of inbred mice were infected intranasally with a neurovirulent clinical isolate of herpes simplex virus type 1. Most mice died without localizing signs of disease in the central nervous system. However, a significant number of SJL mice had a pattern of encephalitis highly reminiscent of that described in humans. To our knowledge, this is the first murine model that faithfully mimics this human disease, and thus it affords the opportunity to study the immunopathogenesis of HSE.
1ID 1991;163 (April)
A Murine Model of Herpes Simplex Encephalitis
721
Table 1. Dose responses of various mouse strains to intranasal infection with herpes simplex virus type 1 (H 129).
Strain
n
SIL A/I
DBAI2
C3H/Hel
AKR
BALB/c
27
C57BL/6 BlO.S
* Mice
35 20
5-10 days
11-13 days
surviving'[
6 5 6 5 6 5 4 6 5 4 4 6 5 6 5 4 6 5 '4 6 6
59 5 100 50 94 100 47 95 100 65 65 100 14 100 93 44 98 100 52 26 25
19 0 0 25 1 0 0 0 0 9 35 0 0 0 0 0 0 0 0 0 0
22 95 0 25 3 0 53 5 0 26 0 0 86 0 7 56 2 0 48 74 75
% mice
sacrificed when moribund. Days = days after inoculation.
t Includes mice that recovered from clinical illness or never displayed clinical signs of disease.
prepared by infecting human embryonic lung (HEL) fibroblasts (cell line MRC-5) with virus at an input multiplicity of 0.01 pfu/cell. The infectivity of the stock was assayed on cultures of African green monkey kidney (Vero) cells (Flow Laboratories, Inglewood, CA) by the plaque assay method using a 2 % methyl cellulose overlay [22]. Results were obtained with virus from a single stock preparation with a titer of 2.25 x 108 pfu/rnl. Clinical and pathologic evaluation. All animals were examined daily for ruffled coats, labored breathing, ataxia, and seizures. Moribund animals were killed and their brains were examined for gross pathologic changes. Brains were sectioned along the midline and fixed in 10 % phosphate-buffered formalin for microscopic examination. Paraffin-embedded brain halves were evenly divided coronally into five blocks, and five coronal sections (6 pm each) were cut from each block. Tissue sections were stained with hematoxylin-eosin (HE) and histologic disease was assessed by determining the presence and distribution of focal inflammatory lesions of hemorrhage and necrosis in the brain. Selected mice were killed by perfusion with saline to determine the distribution of hemorrhage within the brain. Histochemistry. Brains were removed from moribund mice (6-8 animals per strain) and placed in a.C.T. compound (Miles, Elkhart, IN) before freezing. Six serial cryostat sections (6 /Lm each) were cut at 1.5-mm intervals throughout the brain and fixed in acetone (4°C) for 10 s. Rabbit anti-HSV-l and normal rabbit serum (Dako, Santa Barbara, CA) were both absorbed with mouse liver powder (Sigma, St. Louis) and diluted 1:140 before use. The binding of antiHSV-l serum to frozen tissue sectionswas detected by immunoperoxidase staining using the chromogen 3-amino-9-ethylcarbazole. Biotinylated goat anti-rabbit and avidin-conjugated horseradish per-
oxidase were used in accordance with the manufacturer's instruction (Biogenex, San Ramon, CA). Quantitation of infectious virus from CNS tissue. Brain halves were individually homogenized in MEM to yield 10% (wt/vol) homogenates. Plaque assays ofhomogenates (serially diluted) were done in duplicate and virus titers were expressed as plaque-forming units per gram (fresh weight) of tissue. Statistics. Regression lines corresponding to survival time ofvarious HSV-l-infected mice were compared using the t test [23].
Results Clinical patterns of disease in genetically disparate mice infected intranasally with H~l. In the first set of experiments, a viral dose of I (1i pfu of HSV-I was administered to mice of each strain (excluding C.BI7-SCID). Animals from each group were evaluated for clinical and histopathologic evidence of encephalitis. Additional panels of mice of some strains were inoculated with 104 or 105 pfu of HSV-I when it was determined that 106 pfu resulted in 100% mortality before onset of signs of encephalitis. Regardless of mouse strain, the earliest signs of systemic disease were evident 5-7 days after inoculation when affectedanimals had ruffled coats, labored breathing, and hunched posture. The greatest frequency of imminent death (necessitating sacrifice) occurred 5-10 days after inoculation. In strains DBA/2, C3H/He1, AKR, A.SW, BALB/c, C57BL/6, and BIO.S, most animals that would ultimately die of viral infection died during this
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on June 9, 2015
C.BI7-SCID A.SW
54 22 17 12 34 3 15 20 20 23 20 6 7 6 27 9 50 3
% mice moribund *
Virus dose (loglO pfu)
722
Hudson et aI.
Figure 1. Hemorrhagic necrosisof the temporal area (arrow) 11 days after intranasalinoculation of SJL mouse with herpes simplex virus type 1. Bar = 2 mm.
regions of the cerebrum, brain stem, or cerebellum. Tissue sections prepared from surviving mice did not show FERNE lesions . Immunoperoxidase staining for viral antigens in the brains of SJL mice 7 days after inoculation (before the onset of FERNE) revealed that the localization of FERNE·lesions corresponded with the sites of active viral replication (figure 3). The brains of A/J mice that became moribund 11-13 days after inoculation also had eosinophilic necrosis of neurons within the pyriform and entorhinal cortices of the temporal area but lacked the intense inflammatory infiltrate and hemorrhage present in SJL mice . The brains of mice that died 5-10 days after inoculation did not show FERNE. For example, in BALB/c mice (figure 2b) necrosis of individual neural and glial cells was observed scattered throughout cerebrum and brain stem; the cerebellum was unremarkable. Inflammation was modest and diffusely apparent within the temporal area , and a mild cellular infiltrate was present in the adjacent layer of the meninges. However, localizing signs of focal involvement of the brain were not seen.
Quantitationofinfectious virus in the brains ofmoribund miceafterintranasal infection withHSV-J. The striking differences in the histopathology of SJL and other mouse strains led us to determine if there was a difference in the titer of infectious virus in the brains of these animals. Whole brains removed from moribund A/J, DBA/2 , C3H/HeJ, AKR, BALB/c, C57BLl6 (5-10 days after inoculation), or SJL mice (11-13 days after inoculation) were used to quantitate the amount of infectious virus by standard plaque assay technique [22]. Viral titers in whole brain were similar for SJL, C3H/HeJ, AKR, BALB/c, DBA, and C57BLl6 mice (table 2) even though SJL mice became moribund later than the other strains examined. However, SJL mice that became moribund early (5-10 days after inoculation) also had similar viral titers (data not shown) . Viral titrations of brain tissues from mice
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on June 9, 2015
interval (table 1). Many of these animals did not have clinical signs associated with encephalitis, such as ataxia and seizures. By contrast, a significant number of mice of strains A/J and SJL survived a few days longer and did not become moribund until 11-13 days after inoculation . Virtually all of these mice had clinical evidence of encephalitis manifested in the form of ataxia or seizures. While most of these animals died by day 12, a few survived to day 13. Animals that survived beyond day 13 did not die. Survivors included animals that had ocular disease and cutaneous lesions without evidence of encephalitis and those clinically normal throughout the observation period. SJL strain mice have a maturational defect in their immune system that prevents the expression of adult levels of delayedtype hypersensitivity until 10 weeks of age [24]. To determine if the difference in age between SJL mice (10-12 weeks) and the other strains of mice used (8-10 weeks) could account for the patterns of pathology observed, panels of 1O-12-weekold mice (A/J, BALB/c, C57BLl6) were infected with HSV-l and the clinical and histologic pattern of disease was assessed. In these experiments the pattern of pathology observed correlated strictly with mouse strain and was not affected by age difference. Gross pathology. Brains from moribund mice and survivors were examined for gross and microscopic pathologic changes. Gross pathologic changes were not observed in animals that became acutely ill and were sacrificed before day 10 after inoculation. By contrast, SJL mice with clinical signs of encephalitis that became moribund 10-12 days after inoculation showed bilateral softening of the temporal area. Many of these animals also displayed hemorrhagic necrosis of the temporal areas that was usually bilateral, but the intensity of hemorrhage varied considerably from side to side. Perfusion of selected SJL mice showed that hemorrhage, when present, was localized exclusively to the temporal area of the brain (figure 1). There was no evidence of hemorrhage at any other site . Microscopic pathology. HE-stained brain sections from SJL mice had a histopathologic pattern similar to that described in brains of humans with HSE [5-7]. Focal necrotic and inflammatory lesions were seen only within the hippocampus and the entorhinal and pyriform cortexes of the temporal area (figure 2a). In these sites alone , eosinophilic necrosis of neurons, extensive hemorrhage, and intense infiltration with inflammatory cells were observed. The cellular infiltrate comprised largely monocytes and lymphocytes, which formed prominent perivascular cuffs with the lesion. We have designated this histologic pattern of disease "focal entorhinal necrotizing encephalitis" (FERNE) to distinguish it from histologically different forms of encephalitis seen in the brains of other mouse strains (described later) . In other regions of the cerebral cortex of SJL mice, diffuse hypercellularity and "activated" microglial cells were also seen. Importantly, no focal necrotic lesions were observed in other
JID 1991;163 (April)
JID 1991;163 (April)
A Murine Model of Herpes Simplex Encephalitis
723
250 ILm.
.- . .'
with moderate disease yielded only low titers of virus; survivors had none. Two of the five A/J mice assayed showed "-'1O-fold higher amounts of virus than did the other strains assayed. We failed to find an association between the quantity of infectious virus present in the brains of SJL mice, which developed FERNE, and the amount of virus in brains of other mouse strains that also became moribund but had no focal lesions.
tranasal infection with HSV-l were stained for viral antigens by the immunoperoxidase technique. Areas that stained positive for replicating virus were localized within projections of the olfactory pathway. Numerous infected cells were detected in the entorhinal and pyriform cortexes in both SJL (figure 4a) and BALB/c mice (figure 4b) .
Localization ofviral antigens in the brain after intranasal infectionwith HSV-l. Because differences in the quantity of
of the immune system in the pathogenesis of HSE, we also infected C.BI7-SCID mice intranasally with H}' pfu of HSV-l. This strain has a mutation that prevents T and B lymphocytes from generating receptors for antigen-either antibodies or T cell surface receptors. As a consequence, C.B17-SCID mice are deficient in adaptive immunity [25, 26]. All C.BI7-SCID mice infected intranasally with HSV-l 00-
virus could not account for the different histopathologic patterns among SJL and other mouse strains (e.g., BALB/c), we next sought to determine if there was a difference in the distribution of viral antigens in the brains of these animals. Coronal sections from SJL and BALB/c mice 7 days after in-
Patterns ofdisease in C Bl7-SCID mice infected intranasally with HSV-l. Because of interest in the possible involvement
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on June 9, 2015
Figure 2. Coronal sections (hematoxylin-eosin stain) from temporal area of brains from SJL mice with acute disease after intranasal inoculation with herpes simplex virus type 1. A, Focal necrosis of pyriform cortex (enclosed area , arrow) 11 days after infection. In contrast, 7 days after infection BALB/c mice (B) show mild inflammation and meningitis but no focal lesions. Bar =
Hudson et aI.
724
JID 1991;163 (April)
"
...
:
A
1 (HSV-l) antigens in olfactorycenterswithintemporal area of brain from SJL mouse7 daysafter intranasalinoculation with HSV-l. Bar = 500 !-lm .
.-
Table 2. Number of mice with positiveviral culture after intranasalinoculation withherpes simplex virustype 1 (HSV-I) (H129).
B
.No . of mice with HSV-I titers of
IOLI04 104-10 5 105-106 106-107 lOLI08 Strain
n
AKR DBAI2 SJL BALB/c C3H /HeJ C57BLl6 A /J C .B17-SClD
7 5 5 5 6 3 5 5
(p ful g fresh wt)
0 0 1 0 0 0 0 0
2
0 0 0 1
0 0 0
3 4 2 3 .2
2 I 2
2 3 2
2 3
0 0 0 0 0 0 2 1
came infected with this virus. Some animals became moribund as early as 6 days after inoculation; however, a large fraction of animals did not become severely ill and moribund until as late as 11-13 days after inoculation (table 1). When moribund mice from this latter group were sacrificed, the distribution of histopathologic changes observed in their brains was strikingly similar to those observed in BALB/c mouse brains and consisted chiefly of diffuse and scattered necrosis of individual neurons throughout the cerebrum and brain stem (data not shown) . However, the intensity of cellular necrosis rivaled that seen in some A/J mice, which had similar virus titers in the CNS (table 2). Importantly, there were no focal CNS lesions and no evidence oflocalized pathologic changes in any region of their brains. It is unlikely that the extended survival times of C.BI7-SCID mice was due to differences in
Figure 4. Light micrographs of numerous herpes simplex virus type 1 (HSV-l)-infected cel1s (arrows, representative cells)in pyriformarea of brains from SJL (A)and BALBfc (B) mice 7 daysafter intranasal inoculation with HSV-l. Bar = 100 !-lm.
genetic background alone, since a high frequency of these animals survived longer than any of the immunocompetent strains evaluated. Association ofsurvival time with the clinical pattern ofdisease and FERNE. The data obtained from mice infected with viral doses that resulted in the death of at least some of the infected animals (table 1) were used to construct a survival curve (figure 5). This analysis showed two significantly different (P < .002) rates of mortality among the mouse strains infected with HSV-l. The slope of the curve depicting mice that died between 5 and 9 days is most steep (r = 0.99); the slope that described the death rate between days 10 and 13 is less steep (r = 0.93) and the difference between the two slopes is highly significant (P < .002) . Survival times were highly mouse-strain dependent, with SJL and A/J mice representing the largest fraction of animals that died 10-13 days after inoculation (table I). Interestingly, these mice presented the most consistent clinical manifestations of encephalitis, and FERNE was found exclusively in SJL mice that became moribund during this interval. These findings imply that animals infected intranasally with HSV-I develop two
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on June 9, 2015
Figure 3. Immunoperoxidase staining forherpessimplex virus type
A Murine Model of Herpes Simplex Encephalitis
JID 1991;163 (April)
350 ,--u==~:-:------r========;iT'" 20
300 15 01
c:
:~
250
w
~
Z
a:
:J
en
w 10 u,
CD
0
:?: 0
z
0
z
200
5 150
2
--4..............-...._.--tlI--.-----r------'l...-......-
4
6
8
10
12
0
14
Day post-infection
Figure 5. Survival and frequency of encephalitis in various inbred mouse strains after intranasal inoculation with herpes simplex virus type 1 (HSV-l). Mice were infected at virus doses that resulted in survival rates of 3 %-95 %. FERNE = focal entorhinal necrotizing encephalitis.
temporally distinct, and perhaps pathogenically unique, types of disease, only one of which is a possible model of HSE. As anticipated, mortality rates were the highest for C.B17SCID mice. Because of their immunodeficient status, the data from these mice are not included in the survival curve. It is particularly important, however, to examine the actual pattern of survival among these grossly immune incompetent mice. Note that in table 1, 7 of20'C.BI7-SCID mice survived the 5-10-day interval only to die 11-13 days after inoculation. This indicates that immunoincompetent mice can survive a lethal infection with intranasal HSV-l longer than some immunocompetent mice (BALB/c, DBAI2) infected with a viral dose of equal magnitude. Discussion By surveying many genetically disparate inbred strains of mice, we have identified the SJL mouse as a potential model of human HSE. The characteristic features of human HSE that we feel must be reproduced in any suitable animal model include (1) acute onset, (2) usually fatal outcome, (3) localization of pathologic CNS changes preferentially to the inferior temporal area and limbic system, (4) focal inflammatory lesions of necrosis and hemorrhage with this tissue distribution, and (5) occurrence in an apparently immunocompetent host. The disease produced by intranasal infection of SJL with HSV-1 (which we described here) fulfills most of these criteria. A high proportion ofSJL mice died 11-13 days after inocula-
tion after suffering obvious clinical signs of CNS involvement. When their brains were examined microscopically, focal necrotic and hemorrhagic lesions were found, located exclusively within the inferior temporal area. Since the recipients were not exposed to immunosuppressive agents before intranasal infection, and since they were adults (10-12 weeks old), they were fully immunocompetent when virus was instilled. Thus, the criterion of occurrence in an otherwise immunologically normal individual was met. We anticipate that future study of this animal model will shed new light on the pathogenesis of human HSE. Experimental induction of HSV-1 infections of the brains of mice and other laboratory animals has been done many times previously. However, none of the previous experimental strategies produced clinical and histopathologic findings of the type reported here using both an immunologically described model and a proposed route of infection for the human disease [6, 10]. Stroop and Schaefer [27] and Schlitt et al. [28] have both produced focal lesions restricted to the temporal lobes using rabbit models of HSE. However, the use of immunosuppressive agents [27] or the paucity of rabbit immunologic reagents [27, 28] make these models impractical for studying the putative role of the immune response in HSE. Direct intracerebral inoculation of HSV-1 usually causes a diffuse encephalitis that is lethal in 7-10 days [21, 29]. However, McFarland et al. [11] have reported extended survival times and produced focal lesions in the limbic systems of mice after microinjection of a low-virulence strain of HSV-l directly into the hippocampus. Damage to the blood-brain barrier and trauma to surrounding CNS tissue by this artificial method of infection likely has an important impact on the ensuing immune response. Injection of HSV-1 subcutaneously and intraperitonea1ly causes encephalitis in some recipients, depending upon factors such as the number ofinfectious viral particles in the inoculum, the strain of virus, and the genetic constitution of the host [30]. Almost invariably, the pattern of CNS involvement that has been described is diffuse, without localizing or pathologic signs. The intranasal route of infection has also been used previously to cause encephalitis in mice [29, 31, 31a]. However, perhaps because of the viral dose used, or due to the genetic constitution of the recipient, encephalitis of the unique form that occurs in SJL mice, and that resembles human HSE, was not observed. Our findings that moribund SJL and BALB/c mice have comparable viral titers in the brain and the same localization of viral antigens to the primary olfactory areas emphasize the point that host genetic factors playa critical role in determining the pathologic consequence of infection in the brain. Depending upon the dose of virus, a variable proportion of mice of each strain became lethargic, hunched, and moribund 5-10 days after inoculation. Most of these animals had no clinical signs of encephalitis, nor did their brains display FERNE-type lesions. In addition to animals showing this type of disease early after intranasal infection, an equal propor-
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on June 9, 2015
100 -+-
725
726
Hudson et al.
HSV-l. Certain immunologic features of SJL, although their genetic basis may not be well understood, are potentially relevant to development of HSE. For example, SJL mice infected with Theiler's virus develop a demyelinating CNS disease in which an immunopathogenic process appears to intervene as the proximate cause of damage to myelinated nerve fibers [33, 34]. Moreover, among inbred mouse strains, SJL is especially susceptible to the development of experimental autoimmune encephalitis (EAE) induced by immunization with myelin basic protein [35, 36]. Although postulating a relationship between the pathogenesis ofHSE and that of Theiler's demyelination or EAE is premature at this time, the link between an immune response to a viral or tissue antigen and the inflammation that becomes targeted to the brain is similar for all. SJL mice are also of interest because of two other immunologic abnormalities they display. First, Stohlman et al. [24] documented that the ontogenetic appearance of the capacity to develop and express delayed hypersensitivity is delayed in SJL mice relative to other strains. Not until these animals are 10-12 weeks old can they respond to an antigenic challenge with delayed hypersensitivity that rivals the intensity of that observed in other mice. The maturational defect, although still imprecisely understood, appears to be expressed in macrophages [37]. Second, the genome of SJL mice contains a large deletion of genetic material from the locus that encodes the variable part of the {3 chain of the T cell receptor [38]. At this point, neither aspect of the immune system of SJL mice translates directly into an explanation for their susceptibility to HSE. However, since we anticipate that delayed hypersensitivity is likely to be a major effector modality in the expression ofHSE, both defects may be pertinent. As we attempt to understand the immunopathogenesis of HSE in SJL mice, these features will figure prominently in our experimental approach.
Acknowledgment We thank M. Norenberg for assistance in the histologic analysis and Maria Saenz for expert technical assistance.
References 1. Dix RD, Bredesin DE. Vial infections in acquired immunodeficiency syndrome. In: Rosenblum ML, Levy RM, Bredesen DE, eds. AIDS and the nervous system. New York: Raven Press, 1988:221-61. 2. Hotson JR, Pedley TA. Neurological complications of cardiac transplantation. Brain 1976;99:673-94. 3. Pepose JS, HBOOrne LH, Cancilla PA, Foo RY. Concurrent herpes simplex and cytomegalovirus retinitis and encephalitis in the acquired immune deficiency syndrome (AIDS). Ophthalmology 1984;91: 1669-77. 4. Price P, Chernik NL, Horta-Barbosa L, Posner JB. Herpes encephalitis in an anergic patient. Am J Med 1973;54:222-7. 5. Clizer EE, Loannides G. Herpes simplex encephalitis. AJR 1971; 112:273-5. 6. Esiri MM. Herpes simplex encephalitis-an immunohistological study
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on June 9, 2015
tion of mice of some strains (SJL and A/J) became moribund later, 11-13 days after inoculation. Many of these animals developed clear-cut clinical evidence of encephalitis and, when sacrificed, the brains of SJL mice showed the changes typical of FERNE. The brains of A/J mice showed changes in the temporal area characterized by individual cell necrosis, but the intense inflammatory component of FERNE was absent. The presence of high viral titers in some A/J mice suggests that this form of encephalitis may be mediated by direct viral cytopathology, with less of a contribution by the immune response. A third group of animals fits into neither of these two categories; these animals developed either no clinical disease or transient clinical symptoms (usually without CNS signs) . from which they recovered. Histologic evaluation of their brains was unremarkable. These three distinct outcomes after intranasal infection of mice with HSV-1 occurred even though all animals used for these experiments were considered immunocompetent. The importance of this observation is underscored by the outcome of intranasal infection of C.B17-SCID mice in whom adaptive immunity [25, 26] cannot take place because of a genetic defect that precludes their lymphocytes from generating antibodies or lymphocyte surface receptors for antigen. All C.B17-SCID mice infected intranasally with HSV-1 became infected with this virus, becoming moribund as early as day 6 or as late as days 11-13 after inoculation. The death of these latter mice corresponds with the time during which SJL mice developed FERNE. However, the histopathologic changes observed in the brains ofC.B17-SCID mice resembled those observed in brains of BALB/c, not SJL, mice. There were no focal lesions or localized pathologic changes in any region of their brains. This result implicates an immunopathogenic process in the development of FERNE and supports the original hypothesis of Price et al. [4] concerning the participation of the immune system in HSE. We presume that the diffuse pattern of pathologic changes observed in the brains of C.B17SCID mice, without focal necrosis, reflects direct cytopathic effectsof virus in the absence of participation by immune effectors. In addition, while a significant number of C.B17-SCID mice survived beyond day 10, a significant number of "normal" BALB/c mice died 5-10 days after intranasal infection. Although we do not yet know the cause of death in these BALB/c mice, the fact that they died earlier than many similarly infected C.B17-SCID mice implies that the early death of BALB/c mice may also be mediated in part by an immunopathogenic process. Now that HSE can be induced in experimental form in SJL mice, we are interested in understanding the nature of the genetic factors that operate in these mice to render HSE possible. It is known that the major histocompatability complex does not seem to influence susceptibility to HSV infections in mice [32]. That appears true as wellfor HSE, since in our experiment SJL, but not the H-2 congenic B10.S or A.SW strains of mice, developed HSE after intranasal infection with
JID 1991;163 (April)
JID 1991;163 (April)
7.
8. 9.
10. 11.
13.
14. 15.
16.
17.
18.
19.
20. 21.
22. 23.
of the distribution of viral antigens within the brain. J Neurol Sci 1982;4:209-26. Nahmias Al, Whitley RJ, Visintine AN, Taki Y, Alford CA Jr, Collaborative Antiviral Study Group. Herpes simplex virus encephalitis: laboratory evaluations and their diagnostic significance. J Infect Dis 1982;145:829-36. Adams H, Miller D. Herpes simplex encephal itis - a clinical and pathological analysis of 22 cases. Postgrad Med J 1973;49:393-7. Haymaker W, Smith MG, Van Bogaert L, De Chenar C. Pathology of the viral disease in man characterized by nuclear inclusions. In: Field WS, Blatter RL, eds. Viral encephalitis. Springfield, IL: CC Thomas, 1958:95-104. Elizan TS, Maker H, Yahr MD. Neurotransmitter synthesizing enzymes in experimental viral encephalitis. J Neural Trans 1983;57:139-47. McFarland DJ, Sikora E, Hotchin J. The production of focal herpes encephalitis in mice by stereotaxic inoculation of virus. J Neurol Sci 1986;72:307-18. Sckizawa T, Openshaw H. Encephalitis resulting from reactivation of latent herpes simplex virus in mice. J Virol 1986;50:263-6. Kastrukoff LF, Lau AS, Puterman ML. Genetics of natural resistance to herpes simplex virus type 1 latent infection of the peripheral nervous system in mice. J Gen Virol 1986;67:613-21. Lopez C. Genetics of natural resistance to herpesvirus infection in mice. Nature 1975;258:152-3. Allen JE, Doherty Pc. Consequences of a single Ir gene defect for the pathogenesis of lymphocytic choriomeningitis. Immunogenetics 1985;21:581-90. Clatch RJ, Melvold RW, Dal Canto MC, Miller SD, Lipton HL. The Theiler's murine encephalomyelitis virus (TMEV) model for multiple sclerosis shows a strong influence of the murine equivalents of HLA-A, B, and ~. J Neuroimmunol 1987;15:121-35. Lipton HL, Dal Canto MC. Susceptibility of inbred mice to chronic central nervous system infection by Theiler's murine encephalomyelitis virus. Infect Immun 1979;26:369-74. Tabira T. Autoimmune demyelination in the central nervous system. In: Raine CS, ed. Advances in neuroimmunology. Vol. 540. New York: New York Academy of Sciences, 1988:187-201. Touhy VK, Sobel RA, Lees MB. Myelin proteolipid protein-induced experimental allergic encephalomyelitis - variation of disease expression in different strains of mice. '.r Immunol 1988;140:1868-73. Johnson RT. Viral infections of the central nervous system. New York: Raven Press, 1982:135-8. Dix RD, McKendall RR, Barenger RJ. Comparative neurovirulence of herpes simplex virus type 1 strains after peripheral or intracerebral inoculation of BALB/c mice. Infect Immun 1983;40:103-12. Dreesman GR, Benyesh-Melnick M. Spectrum of human cytomegalovirus compliment-fixing antigens. J Immunol 1967;99:1106-14. Glantz SA. Primer of biostatistics. New York: McGraw-Hill, 1981: 180-227.
727
24. Stohlman SA, Matsushima GK, Casteel N, Frelinger JA. The defect in delayed-type hypersensitivity of young adult SJL mice is due to a lack of functional antigen presenting cells. Eur J Immunol 1985;15:913-6. 25. Bosma GC, Custer RP, Bosma MJ. A severecombined immunodeficiency mutation in the mouse. Nature 1983;301:527-30. 26. Dorshkind K, Keller GM, Philips RA, et al. Functional status of cells from lymphoid and myeloid tissues in mice with severe combined immunodeficiency disease. J Immunol 1984;132:1804-8. 27. Stroop WG, Schaefer DC. Production of encephalitis restricted to the temporal lobes by experimental reactivation of herpes simplex virus. J Infect Dis 1986;153:721-31. 28. Schlitt M, Lakerman AD, Wilson ER, et al. A rabbit model of focal herpes simplex encephalitis. J Infect Dis 1986;153:732-5. 29. Anderson JR, Field HJ. The distribution of herpes simplex type 1 antigen in the central nervous system after different routes of inoculation. J Neurol Sci 1983;60:181-95. 30. Dix RD, Mills J. Experimental mouse models of herpes simplex virus infection. In Zak 0, Sande MA, eds. Experimental models in antimicrobial chemotherapy. Vol. 2. London: Academic Press, 1986: 219-57. 31. De Clercq E, Miroslav L. Intranasal challenge of mice with herpes simplex virus: an experimental model for evaluation of the efficacy of antiviral drugs. J Infect Dis 1976;133:A226-36. 3la. TomlinsonAH, Esiri MM. Herpes simplex encephalitis-immunohistological demonstration of spread of virus via olfactory pathways in mice. J Neurol Sci 1983;60:473-84. 32. Lopez C. Resistance to HSV-l in the mouse is governed by two major, independently segregating, non-H-2 loci. Immunogenetics 1980; 11:87-92. 33. Clatch RJ, Lipton HL, Miller SD. Characterization of Theiler's murine encephalomyelitis virus (TMEV)-specific delayed type hypersensitivity responses in TMEV-induced demyelinating disease: correlation with clinical signs. J Immunol 1986;136:920-27. 34. Clatch RJ, Melvold RW, Miller SD, Lipton HL. Theiler's murine encephalomyelitisvirus (TMEV)-induced demyelinatingdisease in mice is influenced by the H-2D region: correlation with TMEV-specific delayed type hypersensitivity. J Immunol 1985;135:1408-14. 35. Kennedy MK, Dal Canto MC, Trooter JL, Miller SD. Specific immune regulation of chronic-relapsing experimental allergic encephalitis in mice. J Immunol 1988;141:2986-93. 36. Pettinelli CB, Fritz RB, Jen Chou CH, McFarlin DE. Encephalitogenic activity of myelin basic protein in the SJL mouse. J Immunol 1982;129:1209-11. 37. Matsushima GK, Stohlman SA. Maturation of the delayed-type hypersensitivity response in SJL mice: absence of effector cell induction. Eur J Immunol 1988;18:1411-16. 38. Huppi KE, D'Hoostelaere LA, Mock BA, et al. T-cell receptor VT(3 genes in natural populations of mice. Immunogenetics 1988;27:51-6.
Downloaded from http://jid.oxfordjournals.org/ at University of Ulster on June 9, 2015
12.
A Murine Model of Herpes Simplex Encephalitis
