Murine retrovirus-induced spongiform encephalopathy: Productive infection of microglia and cerebellar neurons in accelerated CNS disease

Neuron,

Vol. 7, 365-379,

September,

1991, Copyright

0 1991 by Cell Press

Murine Retrovirurlnduced Spongiform Encephalopathy: Productive Infection of Microglia and Cerebellar Neurons in Accelerated CNS Disease William P. lynch,* Stephanie Czub,* Frank J. McAtee,* Stanley F. Hayes,+ and John 1. Portis* *Laboratory of Persistent Viral Diseases +Laboratory of Vectors and Pathogens Rocky Mountain Laboratories National Institute of Allergy and Infectious Disease National Institutes of Health Hamilton, Montana 59840

Summary We have examined the pathological lesions and sites of infection in mice inoculated with a highly neurovirulent recombinant wild mouse ecotropic retrovirus (FrCasE). The spongiform lesions appeared initially as swollen postsynaptic neuronal processes, progressing to swelling in neuronal cell bodies, all in the absence of detectable gliosis. Infection of neurons in regions of vacuolation was not detected. However, high level infection of cerebellar granule neurons was observed in the absence of cytopathology, wherein viral protein was found associated with both axons and dendrites. Infection of ramified and amoeboid microglial cells was associated with cytopathology in the brain stem, and endothelial cell-pericyte infection was found throughout the CNS. No evidence of defective retroviral expression was ob served. These results are consistent with an indirect mechanism of retrovirus-induced neuropathology. Introduction The wild mouse ecotropic retrovirus (CasBrE) induces a noninflammatory spongiform neurodegenerative disease involving caudal segments of the CNS (Andrews and Gardner, 1974; Gardner et al., 1973; Portis, 1990; Swarz et al., 1981). Clinically the disease is characterized by hindlimb paralysis associated with tremor and atrophy of axial skeletal muscles. For field isolates of the virus the incubation period can range from 2 months to as long as 1 year. The pathogenesis of the disease is clearly associated with the capacity of the virus to infect cells within the CNS. However, the nature of the target cells and their role in the neurodegeneration are matters of controversy. It was initially appreciated by Andrews and Gardner (1974) that neurons in the ventral gray matter of the spinal cord exhibited aberrant retrovirus-like particles budding into cytoplasmic vesicles resembling endoplasmic reticulum. However, they noted that these neurons did not manifest ultrastructural evidence of cytopathology. Oldstone et al. (1980) and more recently Sharpe et al. (1990) and Morey and Wiley (1990) have presented evidence that viral proteins are expressed within neurons located in the areas exhibiting neurodegenerative changes. These studies further suggested that vi-

ral replication within neurons might be defective, leading to an accumulation of abnormal amounts of viral gene products (Oldstone et al., 1980; Sharpe et al., 1990). Inherent in these studies was the possibility that virus or viral gene products found within the CNS might not be derived from the inoculated virus, but instead may have originated from endogenous proviruses present in the mouse genome. Neitherthe ultrastructural studies (Andrews and Gardner, 1974; Oldstone et al., 1977; Pitts et al., 1987; Swarz et al., 1981) northe immunohistochemical studies using heterologous anti-virus antisera (Morey and Wiley, 1990; Oldstone et al., 1980; Sharpe et al., 1990) ruled out this possibility. For this reason, the question of whether the wild mouse virus is actually neurotropic has been raised (Pitts et al., 1987), and the possibility of an indirect mechanism responsible for the neuronal cytopathology has been proposed (Pitts et al., 1987; Portis, 1990). Molecular genetic studies of both CasBrE and tsl (a neurovirulent mutant of Moloney murine leukemia virus) have revealed that thedeterminantsfor neurovirulence reside within the env gene (DesGroseiIlers et al., 1984; Szurek et al., 1988; Yuen et al., 1985, 1986). Recently, we constructed a chimeric virus, FrCasE, which contains the env gene of the wild mouse retrovirus and the rest of the genome derived from the FB-29 strain of Friend murine leukemia virus (Portis et al., 1990). Neonatal inoculation of FrCasE results in the accumulation of high levels of viral DNA in the CNS as early as 14days postinoculation and induces a rapid spongiform noninflammatory neurodegenerativedisease involving areas similar to those affected by the wild mouse virus field isolates. Onset of tremulous paralysis is typically evident by 15-16 days postinoculation and occurs in 100% of the mice. The clinical course of the disease takes less than IO days and terminates in death. The rapid onset and highly predictable clinical course, associated with the high levels of CNS infection, make this an ideal system to analyze the relationship between the localization of viral replication and the distribution of lesions. Using highly specific probes, we found that the neurons which exhibit degenerative changes appeared not to be infected by this virus, and neurons that were heavily infected did not exhibit cytopathology. These results suggest that the spongiform neurodegenerative disease is an indirect consequence of viral infection. Results Histopathology Associated with FrCasE Infection Inbred Rocky Mountain White (IRW) mice infected as neonates with FrCasE exhibit clinical signs of neurologic disease by 15-16 days postinoculation. The pathology induced by this virus was found to be exten-

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sive and was characterized by vacuolar degeneration primarily confined to the gray matter of the spinal cord and brain stem, but lesions were also identified in the cerebrum, localized specifically in the deep layers of the motor cortex within the frontoparietal lobe. The overall distribution of pathology observed by 17 days postinoculation is summarized in Figure IA. A characteristic example of the vacuolar changes and cellular composition is illustrated in Figure IB. Here it can be seen that extensive vacuolation was found predominantly within the neuropil. Vacuoles can also be seen within cell bodies themselves; however, this was typically observed in animals with progressive disease examined 17 days postinoculation or later. Degeneration of astrocytes and oligodendrocytes could not be ruled out because the identity of vacuolated cells could not always be determined. Of particular note was the lack of detectable gliosis in regions exhibiting degenerative changes (e.g., Figure IB). This finding was confirmed by examining regions involved in pathology after staining with antibodies identifying astrocytic (glial fibrillary acidic protein [GFAP] expressing) and microglial (F4/80 expressing) cell populations. Comparison of infected and uninfected animals indicated no detectable differences (data not shown). To characterize further the structures in the neuropil that were undergoing degenerative changes, ultrastructural studies were carried out. Figure IC illustratesasectionoftheventral hornofthelumbarspinal cord from an IRW mouse 15 days postinoculation. Here a number of synapses can be seen, and the structures that have swollen can be identified as postsynaptic terminals. Postsynaptic vacuolation was a consistent finding in all areas exhibiting pathology. Presynaptic terminals were unremarkable. This pattern was most readily visualized in mice early in the courseof clinical disease. Later (i.e., 17-24days postinoculation) the vacuoles appeared to have coalesced, and their relationship to synaptic terminals could no longer be clearly identified. It should be noted that in Figure IC there is no evidence of budding virions associated with these vacuoles. Evidence of cell-free viral particles could occasionally be found in the vicinity of swollen postsynaptic terminals, but viral budding into the vacuoles or from the plasma membranes of neuron cell bodies within involved motor areas

Figure

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could not be demonstrated examined.

in any of the eight

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Blood Vessels throughout the CNS Were Infected by FrCasE To address the question of the relationship between the sites of viral replication and the sites of pathology, we used immunohistochemistry, in situ hybridization, and electron microscopy to locate the sites of viral replication. It should be emphasized that both the monoclonal antibody (667) and the antisense probe (WMXB) used in these studies are highly specific forthewild mousevirusanddonot reactwithendogenous viruses (McAtee and Portis, 1985; Portis et al., 1990). Thus, a positive signal indicated the presence of the inoculated virus. Blood vessels throughout the CNS were found to support viral replication at high levels. As can be seen in Figure 2, viral proteins, viral nucleic acid, and abundant viral particles were identified. Virus was found to bud both from endothelial cells and pericytes, and particles were found accumulated predominantly in a subendothelial location as has been found for other murine retroviruses (Swarz et al., 1981; Zachary et al., 1986). However, the widespread distribution of CNS blood vessel infection, irrespective of lesion distribution, suggested that viral infection of these cells was unlikely to play a direct role in the pathogenesis of this disease. Extravascular Spread of FrCasE Both in situ hybridization (Figure 2B) and immunohistochemical studies (Figure 3A) indicated that FrCasE also infected extravascular elements within the CNS. Some of the most obvious envelope-specific staining was seen in highly arborized cells (Figure 3A) reminiscent of astrocytes or ramified microglia (Bignami and Dahl, 1974; Bignami et al., 1972; Perry et al., 1985). Double staining for CFAP and viral envelope protein (Figure3B) showed nocoincident staining, suggesting that the infected cells were not astrocytes. Ultrastructural studies identified viral budding from the plasma membrane of both ramified (Figure 3C) and amoeboid (Figure 3D) microglial cells, indicating that the infection of these cells was productive. Despite the diffuse nature of the vascular infection, infection of the extravascular elements (microglia)

by FrCaG

(A) A sagittal schematic representation of the mouse brain depicting the CNS distribution of spongiform pathology in IRW mice 17 days postinoculation, when clinical symtoms are pronounced. Vacuolation is found in gray matter areas throughout the brain stem and spinal cord as well as the deep layers of the cerebral cortex (shaded areas). (B) A light micrograph of a 1 urn toluidine blue-stained Epon section showing vacuolar degeneration seen in the lateral vestibular nucleus of an IRW mouse 17 days postinoculation (442x). This is typical of spongiform degeneration seen throughout the affected CNS some vacuolation can regions. Vacuolation is generally associated with the neuropil with the sparing of neuronal cell bodies, . however, be seen to have coalesced within cell bodies by this time point (arrows). (C) Electron microscopic examination of vacuolar pathology in the ventral horn of the lumbar spinal cord 15 days postinoculation. Note the association of vacuolation with synaptic endplates (arrows). This pattern of postsynaptic vacuolation was found in all regions of CNS pathology examined by 16 days postinoculation. Bar, 1 urn. (Inset) Presynaptic vesicles are still abundant despite degeneration postsynaptically. Bar, 0.3 urn.

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Figure

2. Vascular

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by FrCaG

16 Days

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(A) lmmunoperoxidase staining with the FrCasE env protein-specific monoclonal antibody 667 of blood vessels located in the frontal cortex, a region devoid of histopathology, and counterstained with Mayer’s hematoxylin. Although vascular staining is prominent, no extravascular staining is apparent in this location (460x). (6) In situ hybridization using WMXB riboprobe in the lateral vestibular nucleus (a region with extensive vacuolation) with bright-field microscopy (Mayer’s hematoxylin counterstained). Grain production was concentrated over blood vessels (BV) and extravascular cells (EV) (350x). Analysis of in situ sections by phase microscopy failed to show specific grain production associated with cells identifiable as neurons or vacuoles (data not shown). (C) Electron micrograph identifying productive infection of both endothelial cells and pericytes. Note viral budding into the lumen of the vessel and into the basement membrane space (arrows). Bar, 1 urn.

was more focal in nature and exhibited a colocalizawith brain stem regions involved in pathology. An example of this is illustrated in Figure 4A, where it can be seen that extravascular staining within the thalamus was associated with spongiform degeneration. However, extravascular env protein was also detected in the absence of pathology in a number of regions of the CNS. For example, Figures 4A and 4E show significant staining within the dentate gyrus of the hippocampus, in cells interior, exterior, and within the granule layer in the absence of pathology. The identity of many of the infected cells in the dentate gyrus remains uncertain, although the position, morphology, and immunochemistry of some were consistent with microglia. The other cells infected in this region may includeoligodendrocytes and/or neurons. Again, in situ hybridization within the hippotion

campus (Figure 4F) shows grain development localized to cells with a distribution consistent with those expressing envelope protein. A detailed electron microscopic study of this region has yet to be undertaken. Other areas of the CNS in which extravascular staining for viral env protein was identified in the absence of pathology included the outer layers of the cerebral cortex, olfactory bulbs, and white matter tracts throughout the CNS, especially in the corpus callosum, cerebellar cortex, and spinal cord (data not shown). Using immunohistochemistry, no evidence for viral envelope protein was detected within neuronal cell bodies in regions of CNS pathology (e.g., thalamus, vestibular nucleus, and spinal cord). Figures 4B-4D are high magnification examples of immunoperoxi-

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(A) lmmunoperoxidase staining for FrCas? env protein in the brain stem (670x). Note the staining associated with blood vessel (arrowheads) as well as highly ramified cells (arrows). (6) To determine whether the ramified cellswere of astrocytic origin, fresh frozen sections (4-6 em) were double stained for FrCasE env protein (red) and GFAP (green). As is shown in this micrograph of cells in the medulla, no colocalization of these two antigens was detected (1300x). This lack of coincident localization was observed throughout the CNS, suggesting that astrocytes were not a target for FrCaG infection. (C) Electron micrograph of FrCasr-infected microglial cells in the lateral vestibular nucleus 17 days postinoculation. These cells are clearly identifiable as ramified microglial cells by their dark-staining cytoplasm, lipofuscin granules, and highly extended rope-like endoplasmic reticulum (Peters et al., 1976). Productive infection is clearly evinced by budding viral particles between microglia (boxed region, [El) and adjacent to surrounding vacuoles (arrows). Note again postsynaptic vacuolation in this field (arrowheads). Bar, 1 pm. (E) Bar, 0.3 pm. (D) Productively infected amoeboid microglial cells could also be identified closely associated with blood vessels with virus accumulating within the surrounding basil lamina. Bar, 1 pm.

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Figure

5. Localization

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(A) lmmunoperoxidase staining (with antibody 667) of the cerebellar cortex of IRW mice infected with FrCas’ and counterstained with methyl green (300x). Note that staining is associated with the input white matter tracts LW), the granule layer (G), and the outer molecular layer (ML) and is excluded from Purkinje neuron cell bodies (P). (6) Uninfected control cerebellum stained as in (A) (300x). No evidence of staining can be seen except from the endogenous peroxidase associated with residual red blood cells not removed during perfusion. (C) In situ hybridization showing specific grain production over the granule cell layer but only weak production over the molecular layer (310 x ). (D) Histological examination of the cerebellar cortex using toluidine blue-stained 1 urn Epon sections from a mouse 17days postinoculation wherein no spongiform degeneration is noted (425x).

dase staining of the thalamus with antibody667 showing viral env protein localization to vascular elements, microglia, and extracellular neuropil but exclusion from the neuronal cell bodies (Figures 48 and 4C). There was no evidence that vacuoles or their mem-

Figure Bodies

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branes contained viral envelope protein. It is of interest that vacuolation in the absence of significant amounts of extravascular staining was also observed (Figure 4D), suggesting that the presence of extravascular viral antigen was not required for the induction

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(A-E) Tissue from fresh frozen sections fixed in formaldehyde, stained with monoclonal antibody667-biotin and peroxidase-conjugated streptavidin, and counterstained with hematoxylin and eosin or methyl green (E). (A) A sagittal section of the diencephalon stained for FrCasE viral env protein (40x). Staining for env protein in the thalamus 0 colocalizes with the spongiform change. H, hippocampus; DC, dentate gyrus. (B-D) Higher magnification views of the lateral thalamic nucleus (240x). (B) Abundant env-specific staining is seen in the vicinity of vacuoles, although neuronal cell bodies show no evidence of env staining and actually appear to exclude the reaction product. This colocalization of extravascular env protein and vacuolar pathology was a common feature of most brain stem regions with pathology, although it was not clearly evident in the spinal cord. (C and D) The association between extravascular env protein staining and pathology was not always present. (C) shows high level staining with only minimal evidence of pathology, whereas (D) shows pathology in the absence of high level extravascular staining. Again in (C), exclusion of stain from neurons is prominent. (E) A high magnification view of the extravascular staining evident in the dentate gyrus of the hippocampus (see [A]), a region that does not express neuronal vacuolation (230x). Some infected cells in this region have been identified as microglia (data not shown); however, the identity of the other infected cells is not yet known, but may include granule neurons (G) or oligodendrocytes based on their localization both within and exterior to the granule layer. (F) In situ hybridization within the dentate gyrus indicates a similar distribution of cells expressing FrCaG nucleic acid and those expressing env protein (110x). C, granule neuron cell bodies.

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of pathology. In addition, in situ hybridization revealed no evidence for viral nucleic acids associated with neuron cell bodies in areas uniformly affected by spongiform degeneration (data not shown). Since the mRNAencodingalloftheviralstructural proteinscontains viral env sequences and would be expected to hybridize to the env-specific probe WMXB, this suggests that there was no defective viral expression at the level of protein synthesis. Finally, ultrastructural studies failed to identify recognizable virions (e.g., Figure IQ, and immunoelectron microscopic studies failed todemonstrateenvprotein within or associated with the vacuoles in the ventral gray matter of the spinal cord, lateral vestibular nuclei, and deep cerebellar nuclei in five paralyzed mice (data not shown). To evaluate whether endogenous retroviral antigens were being expressed in affected neurons, animals were examined immunohistochemically using either a rat anti-env protein monoclonal antibody, 83A25 (which recognizes an epitope common to all known endogenous and most exogenous murine leukemia viruses) (Evans et al., 1990), or the anti-gag protein monoclonal antibodies R161 and RI87 (Chesebro et al., 1983). Viral localization in FrCasE-infected animals using these antibodies showed a staining pattern identical to that observed for antibody 667. Neurons in areas of cytopathology were consistently negative for endogenous viral proteins. These negative results provide further evidence that neurons in areas of pathology were neither productively infected by FrCasE nor expressing endogenous retroviral genes. Neurons of the Cerebellar Cortex Were Productively Infected by FrCasE Theobservation that neuronswhich exhibited cytopathology were not infected suggested that an indirect mechanism may be responsible for this disease. One possibility is that neuronal infection in other areas of the CNS could indirectly affect neuronal function and viability at distant sites. lmmunohistochemical examination of the cerebellar cortex, a major component of the motor system, revealed high level infection of the granule cell layer (Figure 5A) and intense staining of the parallel fibers of the outer molecular layer through all cortical folds of the cerebellum. Additionally, basket and stellate neurons could occasionally be observed to express env protein at the light microscopic level and viral particles when examined with the electron microscope. Although these three neuronal cell types have different functions, they all arise from a common progenitor in the outer granule layer in the postnatal period. Virus-specific staining was also noted in vascular elements and white matter tracts of the cortical folds. The white matter staining resulted from the infection of microglia, which were identifiable with the specific antibody F4/80 (data not shown). No evidence of viral env protein was detected in Purkinje neurons or Bergman glial cells. These results were confirmed using in situ hybridization, (Figure 5C). However, in this case, although silver

grains were deposited over the granule cell layer, there was not a corresponding increase in grains over the outer molecular layer, except for a few cells corresponding to stellate neurons or granule cells, which had yet to migrate to the inner granule layer. This discordance between viral protein and nucleic acid detection suggested that the viral protein seen in the outer molecular layer had been transported from cell bodies in the granule layer. Indeed, this outer parallel fiber layer consists predominantly of the axons of granule neurons. Despite the abundance of viral protein in the cerebellar cortex, no evidence of cytopathology was detected (Figure 5D). Thus, neuronal infection by FrCasE, per se, was not associated with neuronal degeneration. Ultrastructural Localization of FrCasE Replication within the Cerebellar Cortex lmmunoelectron microscopy of the cerebellar cortex using antibody 667 revealed viral envelope protein associated with the plasma membranes of granule cell bodies and the neuronal processes of the glomeruli (Figure 6A). Conventional electron microscopy revealed viral particles budding predominantly from the membranes of neuronal processes associated with the glomeruli (Figure 6B), but occasionally budding from the cell bodies of granule cells was also noted (Figure 66, arrowhead, and inset). In contrast, the envelope protein oriented along parallel fibers in the outer molecular layer (Figure 6C) was not associated with viral particles (Figure 6D). This was consistent with the discordance between the detection of viral protein and viral nucleic acids (Figures 5A and 5C). Cumulatively, theseobservations indicated that abundant viral protein and, in the case of the glomeruli, viral assembly were associated with neuronal structures involved in both afferent and efferent synaptic connections within the cerebellar cortex. Discussion In this report we have used the highly neurovirulent chimeric retrovirus FrCasE inoculated into IRW mice as a model system for the study of spongiform encephalopathy. We have evaluated the rapidly progressing pathology and compared its relationship with the expression of CasBrE-specific nucleic acids, envelope protein, and viral particles. Vacuolar lesions were observed in caudal regions of the CNS, which are generally associated with motor function. This pathological picture is similar to that induced by field isolates of CasBrE (Andrews and Gardner, 1974; Brooks et al., 1980); however, in contrast to the findings of Brooks et al., no pathology was seen in the cerebellar cortex in the FrCasE disease. Fine mapping analysis of thedistribution of pathology indicates that it appears to be restricted specifically to neurons involved in motor function (S. C., unpublished data). Interestingly, vacuolar pathology was reported to initiate in both axons and dendrites for Cas-

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BrE (Swarz et al., 1981), whereas in the rapid FrCasE disease vacuoles appeared to initiate and were most prominent at postsynaptic terminals. These results suggest the synapse as the primary neuronal target. This is not unlike the CNS lesions seen as a result of glutamate-mediated excitotoxicity (for reviews see Choi, 1988; Olney, 1990). It is possible that similar mechanisms may be operating to induce this disease. Significantly, we found no evidence that neurons located in sites of spongiform degeneration were infected with the FrCasE virus, despite using highly specific probes in a variety of techniques, including immunohistochemistry, immmunoelectron microscopy, transmission electron microscopy, and in situ hybridization. A recent report of Kay et al. (1991), who used a similar in situ probe on tissue from mice infected with CasBrE, failed to detect infection of neurons in regions of pathology despite the identification of infected glia and endothelia. Using gp70env-specific monoclonal antibodies and electron microscopy on CNS tissue from CasBrE-infected mice, we have also consistently failed to detect the expression of the inoculated virus within neurons in regions of cytopathology(W. P. L. and J. L. P., unpublished data). Similar studies on the distribution of tsl, a neurovirulent mutant of Moloney murine leukemia virus, failed to detect viral protein in neurons in regions undergoing spongiform degeneration (Zachary et al., 1986; Baszler and Zachary, 1990,199l). The results from these various systems conflict with other recent studies on CasBrE in which viral protein was detected in neurons by immunohistochemistry (Morey and Wiley, 1990; Sharpe et al., 1990). However, because polyclonal anti-whole virus sera was used, it is not clear whether the antibody probes employed in these studies were capable of distinguishing the inoculated virus from endogenous retroviral proteins. In the latter study, Northern blot analysis was used to identify the presence of specific CasBrE transcripts; however, specific localization to neurons by in situ hybridization was not performed, but still a neuronal localization was assumed. The original report of Andrews and Gardner (1974) that identified defective retroviral particles in neurons indicated that this was a rare finding and that the particles did not appear to be associated with regions undergoing degeneration. Given the abundance of endogenous retroviral sequences in the mouse genome it is concievable that the CNS response to stress may also result in the activation of some of these viral genes in neurons. In the present and formerly mentioned studies it appears that specific and significant viral expression is not observed in motor neurons; however, none of these studies can rule out the possibility of viral entry and/or subdetectable expression of viral elements as a factor in retrovirus-induced neurodegenerative disease. Finally, it is not clear whether direct neuronal infection is related to subsequent vacuolation, as infected neurons of the cerebellar cortex did not express pathology. However, it is possible that only mo-

tor neurons are specifically susceptible to the effects of these viruses. An interesting and significant difference between the diseases induced by CasBrE and FrCasE is that gliosis was not seen in the latter. The lack of gliosis did not appear to be due to the acute nature of the disease, as mice given very low doses of the FrCasE virus came down with disease with an extended time course, but still showed no glial response (S. C., unpublished data). In view of the possibility that degenerating neurons may indeed be infected by CasBrE, this difference between the two viruses might be responsible for the lack of a glial response. Another possibility is that although FrCasE induced cytopathology in the dendritic processes of neurons and ultimate loss of motor function, it may not cause neuronal cell death as recognized bythe”neuroimmune system”(Giulian, 1987; Graeber et al., 1988). A lack of neuronal death was suggested by Fraser (1979) after observing severe CNS vacuolation without gliosis in mice infected with the unconventional agent scrapie. In the absence of direct viral infection of neurons it is likely that vacuolation of the postsynaptic processes may be due to infection of other cells associated with these motor regions. Our studies identified several different cell types in the CNS that were infected by FrCasE. Cells of the vascular system, endothelial cells and pericytes, were infected throughoutthe CNS, and their distribution bore no relationship to the location of the spongiform lesions. However, the high level replication of this virus in blood vessels may provide a conduit for viral entry into the CNS. Previous ultrastructural studies on the CasBrE virus revealed extensive budding from the vascular endothelium (Andrews and Gardner, 1974; Pitts et al., 1987; Swarz et al., 1981). It was suggested that the primary mechanism responsible for the vacuolar degeneration may involve disruption of the basal lamina by the budding virus (Pitts et al., 1987). Although this is a credible hypothesis, it is not easily reconciled with the diffuse nature of the vascular infection concomitant with the focal nature of the neuronal cytopathology. It is possible that a breakdown in the blood brain barrier may allow the entry of a toxin affecting a specific subpopulation of neurons. Such a hypothesis may explain why some neurons are spared and why viral antigen associated with the vasculature bore no direct relationship to the sites of pathology. In contrast to the vascular infection by FrCasE, infected ramified and amoeboid microglial cells were concentrated in regions exhibiting spongiform degeneration. This observation raises the possibility that infection of microglial cells by retroviruses could lead to their activation and the subsequent release of neurotoxic substances (Giulian, 1987). The diffusion of such toxins could explain the appearance of pathology in areas with no associated infected microglia. These purported substances would have to have considerable specificity, as many areas with infected microglia show no evidence of neuronal pathology. Evi-

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(A) lmmunoelectron microscopy using antibody 667 and horseradish peroxidase-conjugated goat anti-mouse IgG in the granule layer. Significant reaction product is seen deposited along a granule cell (G) plasma membrane (arrowheads) and along processes (arrows) between the cell body and the mossy fiber glomeruli (MF). Bar, 1 urn. (B) Electron micrographofthegranulecell-mossyfiberinterface showingabundantviral particles within theextracellularspace, budding from cellular processes, and budding from granule cell bodies (inset). Bars, 4 urn.

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(C) immunoelectron microscopy as in (A) of the parallel fiber-Purkinje dendrite interface. Reaction product continuous. Bar, 0.1 urn. (D) Ultrastructural analysis of the parallel fibers. No viral particles could be identified in this region unless they steliate neurons or blood vessels, despite the high level of FrCasr env protein expression. Bar, I brn.

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dence supporting this hypothesis may be emerging given a recent report showing that, in addition to classic inflammatory toxins, human immunodeficiency virus (HIV-I)-infected monocytic cell lines release specific neurotoxins in vitro (Giulian et al., 1990). Furthermore, it was demonstrated that these toxins were inhibitable with the specific N-methyl-o-aspartate receptor antagonist MK801. This group proposed that these specific neurotoxins may play an important role in the induction of HIV-associated encephalopathy. It is of interest that two quite different retroviruses, HIV and the wild mouse virus, have tropism for CNS microglial cells (Vazeux et al., 1978; Watkins et al., 1990). However, unlike acquired immune deficiency syndrome, the spongiform encephalopathy induced by FrCasE exhibits no evidence of inflammation or proliferation of resident microglial cells or astrocytes. The lack of gliosis argues against classic microglial activation as outlined by Giulian andTapscott (1988). Preliminary analyses of microglial markers such as mat-1, vimentin, and nonspecific esterase (see Streit et al., 1988) have revealed no evidence of microglial activation in FrCasE-infected animals. Additionally, in experiments in which infected animals were treated with the N-methyl-o-aspartate inhibitors MK801 or phencyclidine, or the macrophage/microglia inhibitor chloroquine, no alteration in the time course or severity of the disease was observed (W. P. L., unpublished data). Despite these findings, the release of a motor neuron-specific neurotoxin by infected microglia cannot yet be ruled out. Thus, if infected microglial cells are causally involved in the induction of neuronal cytopathology, the mechanism probably does not involve the overproduction of classic inflammatory toxins and/or cytokines. Despite the lack of neuronal infection in areas of neuropathology, wefound high level infection of neurons in the cerebellar cortex in the absence of detectable cytopathology. Interestingly, granule cells make up the major populations of neurons that undergo cell division and migration during the postnatal period. This perinatal cell division also corresponds to the period of CNS susceptibility, i.e., the period in which the virus must be present to cause disease (Czub et al., 1991; Hoffman et al., 1981). The proclivity for retroviruses, in general, to replicate in dividing cells might explain the apparent tropism for granule cells seen in this system. Inoculation of developing chicken embryos with the avian retrovirus RAV-1 results in high level infection of cerebellar granule cells (Ewert et al., 1990); neonatal exposure of some mammals to parvoviruses results in specific destruction of cerebellar granule cells (reviewed in Margolis et al., 1971). It is therefore worth considering the possibility that the high level infection of these cells may be involved in thedramatic acceleration in neurodegeneration induced by FrCasE. The cerebellar cortex functions as a modulator of all motor function, acting through the sole output, Purkinje cells, which are inhibitory on their targets.

The granule cell, on the other hand, is an excitatory interneuron whose major function is to stimulate the Purkinje cell. The FrCasE viral env protein appeared to localize to the axons and dendrites of the granule cells, suggesting the possibility of disrupting normal signal transduction. Alteration of efferent conduction through granule cells, either positively or negatively, could ultimately lead to inappropriate Purkinje cell output and a state of dysregulation in distal motor nuclei, the apparent sites of postsynaptic degeneration. Whether the degeneration could occur as a result of an excitotoxic mechanism, as has been suggested for the motor neuron degeneration seen in a Guamanian form of amyotropic lateral sclerosis (Spencer et al., 1987), or as a necrotic response due to the lack of some trophic factor is purely speculation at this point. It should be pointed out that in studies on the loss of cerebellar granule and/or Purkinje cells (as a result genetic mutations, infection by certain DNA viruses, X-irradiation, or exposure to toxins), evidence of associated vacuolar changes in distal motor nuclei have not been reported (see Sidman, 1968; Margolis et al., 1971). Unlike in the FrCasE disease, however, much of the cytoarchitecture is lost in the genetic cerebellar diseases. In the cerebellar staggerer mutation, neuronal populations of the inferior olivary nucleus (which make climbing fiber contacts with Purkinje cells) are reduced by up to 60% (Shojaeian et al., 1985). Whether this reduction is due to the degeneration of the granule cells, to alteration of Purkinjecells, or to a direct effect of the mutation on inferior olive neurons is not yet clear, but this finding may suggest that alterations in one area of the brain have an effect on other synaptically interconnected areas. The advent and analysis of this rapid model of spongiform encephalopathy have brought further into focus what may be minimally necessary for the induction of spongiform neurodegeneration. Gliosis and neuronal infection in regions of pathology do not appear to be necessary. In addition, defective viral infection appeared not to be required, as viral replication was fully permissive in all infected cell types identified. Furthermore, the initial site of vacuolation appeared postsynaptically on neuronal dendrites, suggesting that they are the primary target for the induction of degenerative changes. Finally, the synaptic degeneration of motor neurons could possibly be caused by a defect in motor system signal transduction as a result of cerebellar neuron infection or the presence of a specific synaptic toxin released by infected microglial cells. Experimental

Procedures

Virus FrCasE is a chimeric murine retroviral constructed as previously detailed (Portis etal., 1990).The3’po/and envgeneswerederived from a molecular clone of CasBrE (clone 15-l), and the rest of the genome, including the long terminal repeats, gag gene, and 5 pal gene, was derived from the molecular clone of a strain of Friend murine leukemia virus, FB29. Viral stocks were prepared from confluently infected Fisher rat embryocells grown on Cyto-

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dex beads (Pharmacia). The titer of viral stock used was 5.4 x 105 ffu/ml. Viral titrations were performed using a focal immunoassay as has been previously reported (Czub et al., 1991). Mice and Inoculations All mice used in this study were IRW (McAtee and Portis, 1985) and were bred and raised at the Rocky Mountain Laboratory animal facilities. Virus was inoculated intraperitoneally (30 ul) into 24-48-hr-old neonates, which were followed for signs of clinical disease beginning 12 days postinoculation. The clinical disease has been described previously (Portis et al., 1990) and consists of initial intention tremor and an abnormal abduction reflex of the hindlimbs when the mice are picked up by the tail. Thisisfollowedwithinadayortwobyhind-andthen byforelimb paralysis and finally convulsive episodes of extensor spasticity, usually initiated when the animals are handled. The first clinical signs are seen 14-16 days postinoculation, and the remaining course runs approximately 10 days, terminating in death. Mice were generally killed 15-18 days postinoculation for the viral localization studies. In all cases, uninoculated age-matched IRW mice were used as negative controls. Histopathology For histopathology studies, tissues were fixed 36 hr in 10% neutral buffered formaldehyde and embedded in paraffin. Sections (4 pm) were stained with hematoxylin and eosin. In some cases semithin sections of Spurr’s embedded material (see below), stained with toluidine blue, were also used for pathologic evaluations. Transmission electron microscopy was performed essentially as described (Sitbon et al., 1986). Specimens were examined and photographed using a Hitachi IIE-I electron microscope at 75 kV. Regions of CNS exhibiting pathology and/or the presence of retroviral env protein at the light microscopic level (eight mice inoculated with FrCasr and five age-matched uninoculated animals) were examined in the electron microscope at ages 1517 days postinoculation for the presence of viral particles and ultrastructural integrity. Double-label Indirect lmmunofluorescence Cryostat sections (4-6 pm), prepared from unfixed, fresh frozen cord and brain, were collected on glass slides, air dried, and fixed in 4% formaldehyde in PBS for 5 min. Slides were washed (3times, 5 min each) in PBS (137 mM NaCI, 2.7 mM KCI, 8.03 mM Na2HP04, 1.47 mM KH2P04 [pH 7.41) and then incubated for 3060 min with biotinylated monoclonal antibody 667 (Portis et al., 1987) (diluted I:100 in RPMI 1640 + 10% fetal bovine serum or medium alone). Slides werewashed (3 times, 5 min) in PBS, incubated with rabbit anti-cow GFAP antibody for 30-60 min, and washed in PBS. The slides were then developed for indirect immunofluorescence by incubation with both tetramethylrhodamine isothiocyanate-labeled streptavidin (diluted 1:2000, Southern Biotechnologies) and FITC-labeled goat anti-rabbit IgC (diluted 1:208, Southern Biotechnologies) for 30 min. All incubations and washings werecarried out at room temperature. Slides wereexamined and photographed using a Leitz Orthoplan fitted with epifluorescence optics. lmmunoperoxidase Tissue preparation and immunohistochemistrywere performed as described by Brown and Farquhar (1984) with minor modifications. Briefly, mice were anesthetized with 2.5% avertin (20 ml/ kg) and perfused through the left ventricle with saline until the effluent ran clear. This was followed by perfusion with paraformaldehyde-lysine-periodate fixative (McClean and :Jakane, 1974). Brains and spinal cords were removed, and the brains were cut into approximately 3 pm thick sagittal or coronal slices and postfixed for 4-6 hr by immersion in room temperature paraformaldehyde-lysine-periodate. Tissue slices were then placed in successive sucrose, PBS solutions of 5%, IO%, 1596, and 20% sucrose at 4°C until the tissue was no longer buoyant. Tissue pieces were rinsed briefly in PBS, blotted, placed in Tissue Tek OCT mounting medium (Miles Inc.), and frozen by immersion in liq-

uid nitrogen. Sections (5-10 urn) were cut using a Reichert Histostat cryotome. Sections were collected on gelatin-coated glass slides and air dried. Slides were rehydrated in PBS, fixed for 5 min in fresh 2% paraformaldehyde, and rinsed for 5 min in three changes of PBS. Primary antibodies (polyclonal antiserum, monoclonal hybridoma supernatants, purified monoclonal antibodies, or purified biotinylated monoclonal antibodies)were placed on the sections and incubated for either 1 hr at 37OC or overnight at 4”C, followed by washing (three times, 10 min each) in PBS (pH 7.2). Specificantibodies used included the following: CasBrE-specific anti-gp70e”” monoclonal antibody 667 (McAtee and Portis, 1985); purified biotinylated 667 (I:100 dilution); broadly reactive antimurine leukemia virus gp7p rat monoclonal antibody 83A25 (Evans et al., 1990); anti-murine leukemia virus p15sag-specific rat monoclonal antibody RI61 and anti-p30sag rat monoclonal antibody RI87 (Chesebro et al., 1983); rat anti-mat-1 monoclonal antibody (Springer et al., 1978); F4/80 macrophagelmicroglialspecific rat monoclonal antibody (Perry et al., 1985), kindly provided by Dr. G. Spangrude (Rocky Mountain Laboratories); and rabbit anti-cow GFAP antiserum (1:200dilution) (DAKO). Secondary antisera were placed on the sections and incubated for 1 hr at 37°C. Secondary antisera used included a I:1000 dilution of horseradish peroxidase-coupled goat anti-mouse IgG (Bio-Rad) oral:5OOdilution of biotin-conjugated rabbit anti-rat IgGfmouse adsorbed, Vector). Sections incubated with biotinylated primary and secondary antibodies were incubated with peroxidaseconjugated streptavidin (Biogenex) for up to 1 hr at 37°C. All sections were rinsed in PBS five times for 5 min each. Sections were fixed in 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) containing 5% sucrose for 60 min, washed (three times, 15 min each) in 0.1 M sodium cacodylate (pH 7.4) plus 7.5% sucrose, and then washed (three times, 15 min each) in 50 mM Tris-HCI (pH 7.4) containing 7.5% sucrose (buffer C). Sections were incubated in 0.2% diaminobenzidine in buffer C for 2-5 min. after which H,02 was added (to a final concentration of 0.01%). The reaction was allowed to proceed for 5-15 min at room temperature to visualize the horseradish peroxidase antibodyantigencomplex.Thesectionswerewashedthreetimesin buffer C and counterstained with methyl green or hematoxylin and eosin. For immunoelectron microscopy, tissues were prepared as for light microscopy except that IO-20 pm sections were collected on gelatin-coated slides. After the final washes in buffer C the samples were postfixed in reduced osmium (Karnovsky, 1971, J. Cell Biol., abstract) in 0.1 M sodium cacodylate (pH 7.4) for 30 min at room temperature, washed briefly in distilled water, dehyc’cated, and embedded in Spurr’s low viscosity resin contained in an inverted gelatin capsule placed over the section. Sections were released from the glass slides using heat. Thin sections were cut, stained, and examined as for transmission electron microscopy described above. In Situ Hybridization In situ hybridization studies were performed as described by Mori et al. (1990). The wild mouse retrovirus env-specific probe WMXB, cloned into pCEM 3 (Promega), was kindly provided by Dr. Markus Czub (Rocky Mountain Laboratory).The plasmid was digested to completion with Hindlll, which cuts adjacent to the Sp6 promotor of the vector. Complementary RNA was synthesized using T7 RNA polymerase. Acknowledgments We would like to thank S. Waliser for excellent technical assistance, Dr. S. Mori for help with the in situ hybridization, and Dr. W. Hickey for enlightening discussions on microglial involvement in neurological disease. This work was supported in part by a National Institutes of Health Intramural Research Training Award fellowship tW. P. L.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be

Neuron 378

hereby marked “advertisement” tion 1734 solely to indicate this

in accordance fact.

Received

June

April

11, 1991; revised

with

18 USC Sec-

11, 1991.

Andrews, J. M., and Gardner, M. B. (1974). Lower motor degeneration associated with type C RNA virus infection neuropathological features. J. Neuropathol. Exp. Neurol. 307.

neuron in mice: 33,285

Baszler,T. V., and Zachary, J. F. (1990). Murine retrovirus-induced spongiform neuronal degeneration parallels resident microglial cell infection: ultrastructural findings. Lab. Invest. 63, 612-623. Baszler, T. V., and Zachary, J. F. (1991). Murine retroviral neurovirulence correlates with an enhanced ability of virus to infect selectively, replicate in and activate resident microglial cells. Am. J. Pathol. 738, 655-671. D. (1974). Astrocyte specific protein in cortex of the newborn rat. Nature 252,

Bignami, A., Eng, L. F., Dahl, D., and Uyeda, C. T. (1972). Localization of glial fibrillary acidic protein in astrocytes by immunofluorescence. Brain Res. 43, 429-435. Brooks, B. R., Swarz, J. R., and Johnson, R. T. (1980). Spongiform polioencephalomyelopathy caused by a murine retrovirus. Lab. Invest. 43, 480-486. Brown, W. J., and Farquhar, M. G. (1984). The mannosephosphate receptor for lysosomal enzymes is concentrated cis golgi cisternae. Cell 36, 295-307.

in

Chesebro, B., Britt, W., Evans, L., Wehrly, K., Nishio, J., and Cloyd, M. (1983). Characterization of monoclonal antibodies reactive with murine leukemia viruses: use in analysis of strains of Friend MCF and Friend ecotropic murine leukemia virus. Virology 727, 134-148 Choi, D. W. (1988). Glutamate neurotoxicity nervous system. Neuron 7, 623-634. Czub, M., Czub, S., McAtee, dependent resistance to murine neurodegeneration results from replication. J. Virol., in press.

and diseases

of the

F., and Portis, J. (1991). Ageretrovirusinduced spongiform CNS-specific restriction of virus

DesGroseillers, L., Barrette, M., and Jolicoeur, P. (1984). Physical mapping of the paralysis-inducing determinant of a wild mouse ecotropic neurotropic virus. J. Virol. 52, 356-363. Evans, L. H., Morrison, R. P., Malik, F. G., Portis, J., and Britt, W. J. (1990). A neutralizable epitope common to the envelope glycoproteins of ecotropic, polytropic, xenotropic and amphotropic murine leukemia viruses. J. Virol. 64, 6176-6183. Ewert, D. L., Steiner, I.,and DuHudaway, J. (1990). In ovo infection with the avian retrovirus RAV-1 leads to persistent infection of the central nervous system. Lab. Invest. 62, 156-162. Fraser, H. (1979). Neuropathology of scrapie: the precision of the lesions and their diversity. In Slow Transmissible Diseases of the Nervous System, Vol. 1, W. J. Hadlow and S. B. Prusiner, eds. (New York: Academic Press), pp. 387-406. Gardner, M. B., Henderson, B. E., Officer, J. E., Rongey, R. W., Parker, J. C., Oliver, C., Estes, J. D., and Huebner, R. J. (1973). A spontaneous lower motor neuron disease apparently caused by indigenous type-C RNA virus in wild mice. J. Natl. Cancer Inst. 51, 1243-1254. Ciulian, D. (1987). Ameboid tion in the central nervous

microglia as effecters of inflammasystem. J. Neurosci. Res. 78,155-171.

Giulian, D., and Tapscott, within the central nervous 358.

M. (1988). lmmunoregulation system. Brain Behav. Immun.

Ciulian, D., Vaca, K., and Noonan, toxins by mononuclear phagocytes 250, 1593-1596. Graeber,

M. B., Streit,

of cells 2, 352-

C. (1990). Secretion of neuroinfected with HIV-I. Science

W. J., and Kreutzberg,

CR3 complement cells. J. Neurosci.

Hoffman, P.M., Ruscetti, S. K., and Morse, H. C. (1981). Pathogenesis of paraylsis and lymphoma associated with a wild mouse retrovirus infection. Age-and dose-related effects in susceptable laboratory mice. J. Neuroimmunol. 1, 275-285.

References

Bignami, A., and Dahl, radial glia in the cerebral 55-56.

otomy of the rat facial nerve leads to increased receptor expre>sion by activated microglial Res. 21, 18-24.

G. W. (1988).

Ax-

Kay, D. G., Gravel, C., Robitaille, Y., and Jolicoeur, P. (1991). Retrovirusinduced spongiform myelencephalopathy in mice: regional distribution of infected target cells and neuronal loss occuring in the absence of viral expression in neurons. Proc. Natl. Acad. Sci. USA 88, 1281-1285. Margolis, C., Kilham, L., and Johnson, R. H. (1971). The parvoviruses and replicating cells: insights into the pathogenesisof cerebellar hypoplasia. In Progress in Neuropathology, Vol. 1, H. M. Zimmerman, ed. (New York: Raven Press), pp. 168-201. McAtee, F. J., and Portis, J. L. (1985). Monoclonal antibodies specific for wild mouse neurotropic retrovirus: detection of comparable levels of virus replication in mouse strains susceptible and resistent to paralytic disease. J. Virol. 56, 1010-1022. McClean, I. W., and Nakane, P. K. (1974). Periodate-lysineparaformaldehyde fixative: a new fixative for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077-1083. Morey, M. localization in moribund

K., and Wiley, C. A. (1990). lmmunohistochemical of the neurotropic ecotropic murine leukemia mice. Virology 778, 104-112.

virus

Mori, S., Wolfinbarger, J. B., Dowling, J. B., Wei, W., and Bloom, M. E. (1990). Simultaneous identification of viral proteins and nucleic acids in cells infected with Aleutian mink disease parvovirus. Microbial. Pathol. 9, 243-253. Oldstone, M. B.A., Lampert, P. W., Lee, S., and Dixon, F. J. (1977). Pathogenesis of the slow disease of the central nervous system associated with WM 1504 E virus. Am. J. Pathol. 88, 193-212. Oldstone, M. B. A., Jensen, F., Dixon, F. J., and Lampert, P. W. (1980). Pathogenesis of the slow disease of the central nervous system associated with thewild mouse virus. II. Role of virus and host gene products. Virology 107, 180-193. Olney, J. W. (1990). Excitotoxicamino disorders. Annu. Rev. Pharmacol.

acidsand neuropsychiatric Toxicol. 30, 47-71.

Perry, V. H., Hume, D.A., and Gordon, S. (1985). Immunohistochemical localization of macrophages and microglias in theadult and developing mouse brain. Neuroscience 75, 313-326. Peters, A., Palay, S. L., and de Webster, H. F. (1976). The Fine Structure of the Nervous System: The Neurons and Supporting Cells (Philadelphia: W. B. Saunders Company). Pitts, 0. M., Powers, J. M., Bilello, J. A.,and Hoffman, P. M. (1987). Ultrastructural changes associated with retroviral replication in the central nervous system capillary endothelial cells. Lab. Invest. 56, 401-409. Portis, Topics

J. L. (1990). Microbial.

Wild mouse retrovirus: Immunol. 2, 11-27.

pathogenesis.

Curr.

Portis, J. L., McAtee, F. J., and Hayes, S. F. (1987). Horizontal transmission of murine retroviruses. 1. Virol. 61, 1037-1044. Portis, 1. L., Czub, S., Garon, C. F., and McAtee, F. J. (1990). Neurodegenerativedisease induced by thewild mouseecotropic retrovirus is markedly accelerated by longterminal repeat andgag-pal sequences from nondefective Friend murine leukemia virus, J. Virol. 64, 1648-1656. Sharpe, A. H., Hunter, J. I., Chassler, P., and Jaenisch, R. (1990). Role of abortive retroviral infection of neurons in spongiform CNS degeneration. Nature 346, 181-183. Shojaeian, H., Delhaye-Bouchaud, N., and Mariani, J. (1985). Decreased number of cells in the inferior olivary nucleus of the developing staggerer mouse. Dev. Brain Res. 27, 141-146. Sidman, R. L. (1968). Development of interneuronal connections in brains of mutant mice. In Physiological and Biochemical Aspects of Nervous Integration, F. D. Carlson, ed. (Englewood Cliffs, New Jersey: Prentice-Hall), pp. 163-193.

Neuron 379

and Microglia

Infection

in Rapid CNS Disease

Sitbon, M., Sola, B., Evans, L., Nishio, j., Hayes, S. F., Nathanson, K., Garon, C. F., and Chesebro, B. (1986). Hemolytic anemia and erythroleukemia, two distinct pathogenic effects of Friend MuLV: mapping of the effects to different regions of the viral genome. Cell 47, 851-859. Spencer, P. S., Nunn, P. B., Hugon, J., Ludolf, A. C., and Ross, S. M. (1987). Guam amyotropic lateral sclerosis-Parkinsonism-dementia linked to a plant excitant neurotoxin. Science 237, 517522. Springer, T., Calfre, C., Secher, D. S., and Milstein, C. (1978). Monoclonal xenogenic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens. Eur. J. Immunol. 8, 539-551. Streit, tional

W. J., Graeber, M. B., and Kreutzberg, plasticity of microglia: a review. Glia

G. W. (1988). 7, 301-307.

Func-

Swarz, j. R., Brooks, B. R., and Johnson, R. T. (1981). Spongiform polioencephalomyelopathy caused by a murine retrovirus. II. Ultrastructural localization of virus replication and spongiform changes in the central nervous system. Neuropathol. Appl. Neurobiol. 7, 365-380. Szurek, P. F., Yuen, P. H., Jerzy, R., and Wong, P. K. Y. (1988). Identification of point mutations in the envelope gene of Moloney murine leukemia virus TB temperature-sensitive paralytogenie mutant tsl: molecular determinants for neurovirulence. J. Viral. 62, 357-360. Vazeux, R., Brousse, N., jarry, A., Henin, D., Marche, C., Vedrenne, C., Mikol, J., Wolff, M., Michon, C., Rosenbaum, W., Bureau, J.-F., Montagnier, L., and Brahic, M. (1978). AIDS subacute encephalitis: identification of HIV-infected cells. Am. J. Pathol. 726, 403-409. Watkins, B.A., Dorn, H. H., Kelly, W. B., Armstrong, R. C., Potts, B. J., Michaels, F., Kufta, C. V., and Dubois-Dalcq, M. (1990). Specific tropism of HIV-1 for microglial cells in primary human brain cultures. Science 249, 549-553. Yuen, P. H., Malehorn, D., Knupp, C., and Wong, P. K. Y. (1985). A l&kilobase-pair fragment in the genome of the tsl mutant of moloney murine leukemia virus TB that is associated with temperature sensitivity, nonprocessing of Pr80enr and paralytogenesis. J. Virol. 54, 364-373. Yuen, P. H., Tzeng, E., Knupp, C., and Wong, P. K. Y. (1986). The neurovirulent determinants of tsl, a paralytogenic mutant of moloney murine leukemia virus TB, are localized in at least two fuctionally distinct regions of the genome. J. Virol. 59, 59-65. Zachary, J. F., Knupp, C. J., and Wong, P. K. Y. (1986). Noninflammatoryspongiform polioencephalomyelopathycaused by a neurotropic temperature-sensitive mutant of Moloney murine leukemia virus TB. Am. J. Pathol. 724, 457-468.