Proc. Nati. Acad. Sci. USAVol. 84, pp. 3866-3870, June 1987Medical Sciences

Transmission in NFS/N mice of the heritable spongiformencephalopathy associated with the gray tremor mutation

(scrapie/murine leukemia virus/neurodegeneration)

PAUL M. HOFFMAN*tt, ROBERT G. ROHWER§, CLAUDIA MACAULEY*, JOHN A. BILELLO*t,JANET W. HARTLEY¶, AND HERBERT C. MORSE III$*Research Service, Veterans Administration Medical Center, Baltimore, MD 21218; tUniversity of Maryland, Baltimore, MD 21201; §Department ofMicrobiology and Immunology, University of North Carolina, Chapel Hill, NC 27514; and sLaboratory of Immunopathology, National Institute of Allergy andInfectious Diseases, Bethesda, MD 20205

Contributed by Richard L. Sidman, January 20, 1987

ABSTRACT It has been shown that the autosomal reces-sive mutation, gray tremor (gt) was associated in the homozy-gous state (gt/gt) with a rapidly fatal spongiform encephalop-athy. Heterozygotes (+/gt) developed mild asymptomaticspongiform brain lesions as did recipient inbred mice inocu-lated with gt/gt brain homogenates, some ofwhom also showedbehavioral abnormalities [Sidman, R. L., Kinney, H. C. &Sweet, H. 0. (1985) Proc. Natl. Acad. Sci. USA 82, 253-2571.In these studies, inbred NFS/N mice inoculated intracerebrallyat birth or as adults with gt/gt or first passage gt brainhomogenates developed a progressive disease characterized bytremor, ataxia, and spasticity. The symptoms were milder andmore slowly progressive than in the gt/gt homozygote, in theparalytic syndrome that followed neonatal inoculation ofNFS/N mice with a wild murine leukemia virus (Cas-Br-MMuLV), or in the rapidly progressive ataxia and terminalbradykinesia that followed scrapie inoculation of NFS/N mice.The noninflammatory spongiform encephalopathy in affectedNFS/N mice resembled that observed in gt/gt homozygotes,+/gt heterozygotes, and asymptomatic recipient inbred miceinoculated with gt/gt brain homogenates. Neither infectiousMuLV nor MuLV proteins were detected in gt/gt brainhomogenates or in affected recipient mouse brains. Scrapie-associated fibrils, readily identifiable in subcellular fractions ofbrains from scrapie-inoculated NFS/N mice, were not detectedin similar brain fractions from NFS/N mice inoculated with gtbrain homogenates. These results confirm and extend thesuggestion that gt spongiform encephalopathy has both heri-table and transmissible properties. Moreover, the transmissi-ble agent of gt disease differs from both Cas-Br-M MuLV andscrapie in its disease-inducing properties in NFS/N mice. Thecapacity of NFS/N mice to express transmitted gt encephalop-athy as clinical disease, to rapidly express Cas-Br-M MuLVspongiform encephalomyelopathy, and to develop mouse-adapted scrapie after a very short incubation time suggest adistinct sensitivity of NFS/N mice to transmissible spongiformencephalopathy.

Transmissible spongiform encephalopathies can be inducedin mice by inoculation of the agents causing scrapie andCreutzfeldt-Jakob disease (reviewed in ref. 1) and by neo-natal exposure to a number of ecotropic murine leukemiaviruses (MuLVs) (2). A spontaneously occurring spongiformencephalopathy associated with the gray tremor (gt) autoso-mal recessive mutation in mice has been described, andevidence for its transmissibility has been reported (3, 4).While the identity of the agent responsible for gt spongiformencephalopathy is unknown, similarities to the spongiform

encephalopathies induced by both scrapie-like agents andMuLV have been noted (4).Host genetic factors influence the expression, latency, and

severity of spongiform encephalopathies induced by eitherMuLV (5, 6) or scrapie-like agents (1) and play a significantrole in determining the expression of transmissible gt spong-iform encephalopathy (3). Inbred NFS/N mice are remark-ably susceptible to both the wild MuLV (Cas-Br-M MuLV)-(6) and temperature-sensitive Moloney MuLV (tsMo-BA-IMuLV)-induced spongiform encephalopathy (7). In thisstudy, we compared the diseases produced by inoculation ofneonatal and adult NFS/N mice with primary gt/gt brainhomogenates, brain homogenates from mice inoculated withgt brain homogenates, mouse-adapted scrapie (Comptonstrain), and Cas-Br-M MuLV. Differences in clinical symp-toms and disease progression, the absence of infectiousMuLV and MuLV proteins, and failure to detect scrapie-associated fibrils (SAF) (8, 9) show thai the agent responsiblefor gt spongiform encephalopathy in mice behaves differentlythan both MuLV and the scrapie agent in NFS/N mice.

MATERIALS AND METHODSOrigin and Preparation of Inocula. Mice homozygous for

the gt/gt mutation were obtained from The Jackson Labo-ratory (3). Two severely affected mice were sacrificed at 6weeks of age, and 10% (wt/vol) gt/gt brain homogenateswere prepared and used for immediate inoculation and virusisolation studies.Frozen brain and spinal cords from two C3HeB/FeJ mice,

and a BALB/cBY mouse, all of which demonstrated mildbrain stem spongiform pathology 23 months after neonatalinoculation with a5% (wt/vol) brain homogenate from a gt/gtrmiouse were obtained from R. Sidman (Harvard University).An additional brain was obtained from an NFS/N mouse thatdemonstrated spongiform encephalopathy and was tremu-lous and ataxic 12 months after inoculation with a 5%(wt/vol) brain homogenate from a moribund gt/gt mouse.

First passage (P1) gt brains were homogenized inDulbecco's PBS (pH 7.4), clarified by centrifugation, andstored at -70°C as 10% (wt/vol) homogenates prior toinoculation. Brain homogenates from the C3HeB/FeJ andBALB/cBY mice were pooled while the homogenate fromthe NFS/N mouse was used as a separate inoculum. A 10%(wt/vol) brain homogenate from a 12-month-old normalNFS/N mouse was similarly prepared and used as a controlinoculum.

Abbreviations: SAF, scrapie-associated fibrils; MuLV, murine leu-kemia virus; i.c., intracerebrally.1To whom reprint requests should be sent at: Research Service (151),Veterans Administration Medical Center, 3900 Loch Raven Bou-levard, Baltimore, MD 21218.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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An early passage of the Compton strain of mouse-adaptedscrapie, obtained from Alan Dickinson (Edinburgh, Scot-land) was passaged once in C57BL/10/NCr mice. A singlebrain from a mouse terminally ill with scrapie was homoge-nized in PBS, clarified by brief centrifugation and stored at-700C as a 10% (wt/vol) homogenate prior to inoculation.Cas-Br-M MuLV was propagated on SC-1 cells and titered

by XC assay as described (10). Supernatant pools used forinoculation had a titer of 105.2 plaque-forming units/ml.

Inoculation and Clinical Evaluation. NFS/NCr, BALB/cAnNCr, and C57BL/10/NCr mice were obtained from theAnimal Genetics and Production Branch, National CancerInstitute, Frederick, MD, through a VA-NCI InteragencyAgreement. Pregnant NFS/N mice were housed separately.Mice (0-2 days old) were inoculated intracerebrally (i.c.)with 0.01-0.03 ml of 10% (wt/vol) normal NFS/N brainhomogenate, 10% (wt/vol) gt/gt or gt (P1) brain homogenate,10% (wt/vol) scrapie infected-mouse brain homogenate, orsupernatant from Cas-Br-M MuLV-infected SC-1 cells. Micewere weaned at 3-4 weeks, separated by sex, and housed at3-5 mice per cage. Adult NFS/N, BALB/c, and C57BL/10mice (6-12 weeks old) were inoculated under light anesthesiathrough the orbit with 0.03 ml of 10%o (wt/vol) gt (P1) brainhomogenate, 10% (wt/vol) scrapie brain homogenate, and10% (wt/vol) normal NFS/N brain homogenate. All mice inthis study were housed in filter-bonneted cages in the sameroom with uninoculated sentinel mice, that remained clini-cally and histologically normal.Mice were examined weekly for neurologic disease begin-

ning 1 week after inoculation and every 3-4 days beginning4 weeks after inoculation. To control for possible observerbias, the clinical examinations were conducted blind. NFS/Nmice inoculated with normal brain and uninoculated controlNFS/N mice of similar age were included in each evaluation.Mice were considered symptomatic when unequivocal trem-ulousness of the hind limbs or trunk was present on two ormore successive examinations. Agreement between observ-ers was >90%. A third examiner, unfamiliar with neurologicsigns in NFS/N mice, evaluated symptomatic and asympto-matic mice 84 days after inoculation. Concordance forneurologic disease with the previous observers was >95%.

Histopathology and Immunohistochemistry. Mice were sac-rificed by cervical dislocation, and their brains were removedand immersion-fixed in 10% (vol/vol) neutral-buffered for-malin. Sections of cortex, hippocampus, cerebellum, brainstem, and spinal cord were stained with hemotoxylin andeosin. Immunohistochemical studies were also performed onbrain and spleen samples from mice perfused with 10%(vol/vol) neutral-buffered formalin or a perfusate containingsodium periodate/lysine monohydrochloride/paraformalde-hyde/glutaraldehyde (PLPG) (11). The tissues were immer-sion-fixed for an additional 6 hr in PLPG or 24 hr in formalin,washed, dehydrated, and embedded in paraffin.The immunohistochemical techniques used for these stud-

ies will be described in detail elsewhere (E. E. Cimino andP.M.H., unpublished observations). Briefly, deparaffinizedsections were washed with PBS, blocked for endogenousperoxidase with methanol containing 0.1% hydrogen perox-ide, and treated with 3% (vol/vol) normal rabbit serum. Anincubation with primary antiserum (i.e., goat anti-RauscherMuLV p30 or gp7O) was followed by reaction with biotin-labeled rabbit anti-goat IgG (Vector Laboratories, Burlin-game, CA) and avidin-conjugated horseradish peroxidase(Vector Laboratories). The reaction product was developedby incubation with 3,3'-diaminobenzidine tetrahydrochloride(Sigma) and 0.01% hydrogen peroxide.

Identification of MuLV p3O and gp7O by Immunoblotting.Spleens, brains, and spinal cords were removed from inoc-ulated and control mice and homogenized in 0.01 M potas-sium phosphate (pH 7.4) containing 1% Triton X-100 for

detection of viral proteins. Samples containing 300-400 gg ofprotein were analyzed on 3-mm thick, 18-cm long 12.5%NaDodSO4/polyacrylamide slab gels using the discontinuousTris glycine buffer system of Laemmli (12). Proteins weretransferred to nitrocellulose at 40C for 16 hr at 19 V inTris/glycine/methanol buffer, essentially as described byBurnette (13). Transfer of proteins between 3 and 200 kDawas monitored by prestained molecular size markers (Be-thesda Research Laboratories) on the same gel as tissuehomogenates. Unoccupied protein binding sites on the nitro-cellulose membrane were blocked by incubating the mem-brane for at least 8 hr in 3% (vol/vol) fish scale gelatin(Nordlund Laboratories, Newark, NJ). Viral proteins werevisualized after reaction with primary antisera to purifiedRauscher gp7O and p30 followed by horseradish peroxidase-coupled rabbit anti-goat IgG and 4-chloro-1-naphthol chro-magen. Alternatively, viral proteins were detected byautoradiography after reaction with primary goat antibodyand radioiodinated protein A.

Identification of SAF. SAF were prepared by a modificationof the original method (8) that has been used successfully todemonstrate SAF in a wide variety of agent-host combina-tions (9). Single brains were homogenized in 0.32 M sucrose,1 mM MgCl2, 0.5 mM CaCl2, 1 mM NaHCO3 with eightstrokes of a 15-ml Teflon-glass homogenizer, then diluted to20 ml in the same buffer, and centrifuged at 1200 x g for 10min at 40C. The supernatant was saved, and the pellet wasresuspended in 20 ml of the same buffer and recentrifuged at850 x g for 10 min at 40C. The combined supernatants werecentrifuged again at 850 x g for 10 min at 40C, transferred toa fresh tube, and centrifuged again at 13,000 x g for 10 minat 4°C. The pellet of the 13,000 x g spin was resuspended bytrituration in 2 ml of 0.1 M Tris-Cl, pH 7.5, followed by theaddition of 2 ml of 5% (vol/vol) sarkosyl in Tris just prior toloading all 4 ml onto a step gradient of 4 ml of 2 M sucroseand 4 ml of 1.4 M sucrose in Tris. The gradients werecentrifuged overnight at 30,000 rpm in an SW41 rotor at 60C.SAF were collected from the 2 M/1.4 M sucrose boundary,diluted 1:5 and 1:10 (vol/vol) with water, applied to glow-discharged, carbon-coated grids, and stained with freshlyprepared 2% (wt/vol) phosphotungstic acid for electronmicroscopic visualization.

RESULTSNeurologic Disease. Seventeen newborn NFS/N mice were

inoculated (i.c.) with 0.01 ml of clarified 10% (wt/vol) brainhomogenate prepared from gt/gt brains and observed forsigns of neurologic disease. All inoculated mice developed aneurologic disease characterized by tremulousness that pro-gressed to marked ataxia, weakness, and spasticity, moreoften involving the hind than forelimbs. ]Deaths related tosevere neurologic disease were observed at 258-580 daysafter injection in 16 of the 17 mice. One mouse died with ahepatic fibrosarcoma.

Forty-six newborn NFS/N mice were inoculated (i.c.) with0.01-0.03 ml of clarified 10% (wt/vol) gt (P1) brain homog-enate. Mild tremulousness occurred in all mice within 60 daysof inoculation, and a slowly progressive syndrome of increas-ing tremulousness, ataxia, and terminal bradykinesia andspasticity developed over 360-440 days (Fig. 1). Eight adultNFS/N mice (12-18 weeks old) inoculated (i.c.) with thissame gt (P1) homogenate developed tremulousness between98 and 150 days after inoculation and became terminally illbetween 340 and 438 days after injection (Fig. 1). Three of 4male NFS/N mice inoculated as adults and 2 of 4 inoculatedas neonates, developed marked obesity during the course ofdisease. Obesity was not observed among 40 female NFS/Nmice.

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.WL

Scrapic(adult)

(nScrapietT(neonatal) n

gt (neonatal)

gt (adult)

0 40 80 120 160 200 240 280 320 360 400 440

DAYS POST-INOCULATIONFIG. 1. The onset and progression of neurologic disease in NFS/N mice after inoculation with gt (P1) brain homogenate, scrapie (Compton

strain), and Cas-Br-M MuLV. The mean and standard deviation for onset and duration are shown. NFS/N mice inoculated with gt (P1) brainhomogenate were sacrificed at 406 or 440 days when all mice were severely clinically ill.

Scrapie-inoculated neonatal and adult NFS/N mice initial-ly developed tremulousness, followed by prominent ataxia,bradykinesia, and in terminal illness, stiff tails and priapism(males). The onset of clinical disease began 41-74 days afterinoculation in 16 NFS/N mice inoculated as adults; death ormoribund status developed 130-181 days after inoculation(Fig. I and Table 1). A similar syndrome began 78-99 daysfollowing neonatal inoculation of scrapie in 20 NFS/N miceand resulted in death or moribund status 139-160 days afterinoculation (Fig. 1). None of the scrapie-inoculated NFS/Nmice developed obesity during the course of their disease.C57BL/1ON and BALB/cAnN mice inoculated with scrapieat 6-12 weeks of age developed similar clinical syndromeswith onsets at 140-150 days and death at 173-226 days oronsets at 122-133 days after inoculation and death at 181-235days (Table 1).Seven NFS/N mice inoculated neonatally with Cas-Br-M

MuLV developed tremulousness, hind limb weakness, andterminal paralysis by 42-56 days after inoculation and diedwithin 74-120 days after inoculation (Fig. 1). Adult miceinoculated with Cas-Br-M MuLV develop an immune re-sponse against Cas-Br-M MuLV but do not develop neuro-logic disease (10).

Histopathology. Mice inoculated with gt (P1) brain homog-enate demonstrated mild spongiform vacuolation within theneuropil of the brain stem and hippocampus at 5-6 weeksafter inoculation. Vacuolation became more extensive duringthe course of disease, but gliosis was mild and neuronalvacuolation was rarely observed (Table 2). Similar findingswere observed in gt/gt brain homogenate-inoculated miceTable 1. Clinical expression of scrapie in three inbredmouse strains

Symptom onset, Disease duration,Strain days after inoculation days*

NFS/N 65 + 3 93 ± 4BALB/cAnN 127 ± 1 92 ± 6C57BL/1ON 147 + 4 45 + 5

Sixteen NFS/N, 9 BALB/c, and 11 C57BL/10 mice were inocu-lated intraorbitally with scrapie (Compton strain) at 6-12 weeks ofage. Symptom onset is presented as days (mean ± SEM) followinginoculation when tremulousness and ruffled fur or bradykinesia firstbecame apparent.*Mice were sacrificed when moribund in some cases.

examined when severely affected at 250-360 days afterinoculation.

Scrapie-infected NFS/N mice demonstrated vacuolationand gliosis in the hippocampus by 4-5 weeks after inocula-tion. Increasing vacuolation and marked gliosis involvingdeep cortical gray matter, striatum, and thalamus occurred asthe disease progressed. Cerebellar and brain stem gray matterand the spinal cord were less affected.Cas-Br-M MuLV-induced vacuolation and gliosis were

apparent at 3 weeks after inoculation and became moreextensive and widely distributed as the disease progressed.Vacuolation in the neuropil, at the gray-white junction, andwithin the cell bodies ofastrocytes and neurons was observedin the lower spinal cord, deep cerebellar nuclei, and brainstem. Gliosis was prominent in the areas of neuropil vacuola-tion. The neuronal population in these areas was reduced,and many of the remaining neurons were vacuolated (Table2).

Detection of Infectious MuLV, MuLV Proteins, and SAF.Aliquots (0.1 ml) of the original extracts from gt/gt brainswere tested in tissue culture for infectious ecotropic, xeno-tropic, and mink cell focus-inducing MuLV. Mitomycin-treated cell suspensions prepared from the spleens of thesemice and from two +/gt heterozygous mice were also testedin infectious center assays for MuLV of these classes. Noinfectious MuLV of any class was detected in extracts fromgt/gt or +/gt brains. Xenotropic viruses were recoveredfrom both gt/gt spleens after two blind passages of mink cellsin culture but no ecotropic (XC+) or mink cell focus-inducingviruses were detected. Immunoblots of brain homogenatesprepared from affected NFS/N mice inoculated with gt (P1)brain homogenate showed no accumulation of gp7O or p30proteins, and no evidence of MuLV p30 or gp7O could bedetected in the brains or spleens of affected mice by immu-nohistochemical techniques. In agreement with an earlierstudy (14), no MuLV p30 or gp7O could be identified in thespleen or brains of scrapie-inoculated mice. In contrast,MuLV p30 and gp7O were easily detected on immunoblots ofbrain and spleen extracts from Cas-Br-M MuLV-inoculatedparalyzed mice (Fig. 2). Similarly, MuLV p30 and gp7Odemonstrated a perivascular distribution by immunohisto-chemical techniques in brain stem, cerebellar folia, and spinalcords of Cas-Br-M MuLV-infected NFS/N mice (Table 2).SAF were detected by electron microscopy in concentratedbrain homogenates from six scrapie-infected mice sacrificed

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Table 2. Spongiform encephalomyelopathy in NFS/N mice

Histopathology

Inoculum Vacuolation Gliosis Neuron loss MuLV proteins* SAFt

Cas-Br-M MuLV + + + +Scrapie + + + - +gt brain + + - - -

Sections were scored as follows: Moderate numbers (+) of vacuoles were present in the neuropil;reactive glial were present in moderate (+) or minimal (±) numbers; and neurons were degeneratingand had been replaced (+) by glial cells; or there was no loss of neurons (-).*MuLV gp7O and p30 were detected in brain and spleen homogenates of Cas-Br-M MuLV-inoculatedmice by immunoblotting and in fixed brain and spleen by an immunoperoxidase technique (+). MuLVgp70 and p30 could not be detected in scrapie or gt brains (-).tSAF was detected in subcellular fractions of brain from six scrapie-infected mice (+) but not from fivegt brain-inoculated, three normal NFS/N brain-inoculated, and five Cas-Br-M MuLV-infected mice(-)-

60-130 days after inoculation. SAF were not detected in brainhomogenates from five Cas-Br-M MuLV-infected, five gt(P1) brain homogenate, or three NFS/N brain homogenate-inoculated mice (Table 2).

DISCUSSIONThe autosomal recessive gt mutation is associated with arapidly progressive and severe spongiform encephalopathy inhomozygous (gt/gt) mice and mild asymptomatic spongiformlesions in heterozygous (+/gt) mice (3, 4). Initial transmis-sion attempts resulted in spongiform pathology but no con-sistent clinical disease in recipient mice inoculated with gt/gtbrain homogenates (3). These studies suggested that theexpression of gt spongiform encephalopathy was determinedby the interaction of genetic factors and a transmissible agent(3). The studies reported here strongly support that sugges-tion and demonstrate that inbred NFS/N mice consistentlydevelop a slowly progressive neurodegenerative syndromeassociated with spongiform pathology after inoculation (i.c.)with gt/gt and gt (P1) brain homogenates.The clinical syndrome in NFS/N recipient mice is initially

characterized by tremulousness beginning 60 days afterinoculation and progressing slowly over 300-400 days tomarked ataxia, terminal bradykinesia, and spasticity. Thesyndrome was less severe than the whole body tremor,seizures, and death by 90 days of age that occurs in gt/gthomozygotes, but was more clinically apparent than in themild nonprogressive tremulousness reported in mice inocu-lated with gt/gt brain homogenate (3). Affected NFS/N

m a b c d e f

it'9 Pr67 gag

_W _0 *- P30

FIG. 2. Brain homogenates 10% (wt/vol) from two NFS/N miceinoculated with scrapie (lanes a and b), gt (P1) brain homogenate(lanes c and d), or Cas-Br-M MuLV [lanes e and f (1:10 dilution)] andradiolabeled molecular size markers of25.7, 43, 68, 97.4, and 200 kDa(lane m) were electrophoresed and transferred to nitrocellulose. TheMuLV core protein (p30) and its precursor polypeptide (Pr67Pg)were identified on an immunoblot by reaction with a broadly reactivegoat anti-Rauscher MuLV p30 serum and radioiodinated protein A.

recipient mice showed mild spongiform lesions in the graymatter of the brain stem, deep layers of the cerebral cortex,and hippocampus beginning 5-6 weeks after inoculation. Thedistribution of lesions was thus more widespread than thatdescribed in other recipient strains or in the +/gt heterozy-gotes (3, 4), but significant gliosis and neuronal vacuolationand dropout were again not apparent (3, 4).The clinical presentation and histopathology ofgt/gt and gt

(P1) brain homogenate-induced spongiform encephalopathyin NFS/N mice differed significantly from both scrapie- andCas-Br-M MuLV-induced spongiform encephalomyelopathyin NFS/N mice (Fig. 1 and Table 2). Scrapie-inoculated micehad more severe spongiform lesions beginning in the hip-pocampus and progressing to thalamus, brain stem, andcerebellum. Moderate to marked gliosis accompanied thespongiform lesions producing status spongiosis (15) in someareas where neuronal dropout was also apparent. Cas-Br-MMuLV spongiform encephalomyelopathy had a more re-stricted distribution of lesions involving spinal cord graymatter, the gray-white junction, brain stem, and cerebellum(16). The vacuoles, which tended to be larger and morecoalescent than in either scrapie or gt encephalopathy, wereaccompanied by gliosis and neuronal dropout. The prominentgliosis and neuronal dropout in both scrapie and Cas-Br-MMuLV spongiform encephalopathies could account for theirmore severe and rapidly progressive clinical courses. Theobesity that occurred among gt (P1) brain homogenate-inoculated NFS/N males was not explained by an increasedquantity or intensity of vacuolating hypothalamic pathologyas was reported for some scrapie agent-mouse strain com-binations (17).The data presented here provide further evidence that the

transmissible agent from gt brain responsible for this clini-cally and histologically distinct disease behaves differentlythan MuLV known to induce spongiform encephalopathy andscrapie in NFS/N mice. Replication-competent MuLV werenot detected in tissue culture assays ofprimary extracts fromgt/gt brains, and immunoblotting and histochemical studiesof brains from clinically-affected gt/gt- and gt (P1)-inoculat-ed mice showed no accumulation of MuLV-related gp70 orp30 proteins. In addition, SAF could not be detected insubcellular fractions ofbrain homogenates from NFS/N miceinjected with gt (P1) extracts. These results are in keepingwith the earlier findings that budding virions were notdetected by electron microscopic studies of gt/gt brains andthat SAFs were absent from these tissues (3). These findingsdo not rule out the possibility that gt mutation is associatedwith the activation of a scrapie-like agent that has propertiessomewhat different from the well-characterized Comptonstrain of scrapie. Scrapie and Creutzfeldt-Jakob strainsproduce various degrees and distributions of spongiosis andgliosis (1), and SAF are less-easily demonstrated in some hoststrains than in others (18).

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Our results also indicate a striking susceptibility ofNFS/Nmice to agents that induce spongiform encephalomyelopa-thies. First, the latency for clinical disease in mice injectedwith the Compton strain of scrapie was significantly shorterthan for C57BL/10 or BALB/c mice infected with the sameagent. Second, NFS/N mice injected with gt brain homog-enates developed clinical as well as histologically evidentdisease, whereas histologic changes but no clear-cut clinicalsymptoms were observed in studies of gt/gt homogenate-inoculated C3HeB/FeJ and BALB/cBY mice (3). Finally,NFS/N mice have been shown to be more susceptible thanseveral other strains to the paralytic effects of Cas-Br-MMuLV (6). This strain may thus prove to be useful in defininghost genetic factors that influence the development of spong-iform encephalopathies.We thank Eugene Cimino and John Mummaw for technical support

and Cathy Bryson for manuscript preparation. This work wassponsored in part by the Veterans Administration.

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Neurol. 45, 108-126.

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