The distribution of challenge virus standard rabies virus versus skunk street rabies virus in the brains of experimentally infected rabid skunks

Summary The proposal that the bizarre behavioral changes which occur during rabies infection are due to selective infection of limbic system neurons was further studied in skunks (a species important in naturally occurring disease). A detailed immunohistochemical study of brains of skunks experimentally infected with either Challenge virus standard (CVS) or street rabies virus revealed only trace amounts of viral antigen in many limbic system neurons and marked differences in viral distribution between street and CVS virus. These data were collected during early stage rabies when behavioral changes occur. Areas which contained heavy accumulations of street rabies virus but low amounts of CVS rabies virus were the neuronal perikarya and processes of the dorsal motor nucleus of the vagus, midbrain raphe, hypoglossal and red nuclei. In contrast, large accumulations of CVS virus were found in the Purkinje cells of the cerebellum, the habenular nuclei and in pyramidal cells throughout the cerebral cortex, while corresponding areas in all street virus-infected skunks contained minimal antigen. These findings were very consistent for animals of the same experimental group and between skunks inoculated both intramuscularly and intranasally with skunk street virus. Skunks inoculated intramuscularly with CVS rabies virus failed to develop rabies. Since, in this model, street virus infection generally produces furious rabies and CVS infection results in dumb rabies, we speculate that the behavioral changes which occur in these two different clinical syndromes are due to the heavy and specific accumulation of virus in different regions of the CNS. These results show that regions other than those of the limbic system may also be involved in the pathogenesis of behavior changes in rabid animals.Supported by an MRC fellowship (NLS)     

Rabies: Spongiform lesions in the brain

Summary Striped skunks (Mephitis mephitis) experimentally infected with street rabies virus developed spongiform lesions that light- and electron-microscopically were indistinguishable from those found in the transmissible spongiform encephalopathies of man and animals. These previously unreported lesions were also detected in naturally occurring cases of rabies. The spongiform lesions consisted of round or oval vacuoles in the neuropil, rarely in neuronal perikarya. The most severely affected areas were the thalamus and cerebral cortex. The implications of this finding include similarities in the pathogenetic mechanisms of rabies and the traditional spongiform encephalopathies and the possibility of lesion variation due to differences in rabies viral strains. The spongiform lesions of rabies will require consideration in differential diagnosis.     

Topographic distribution of scrapie amyloid-immunoreactive plaques in chronic wasting disease in captive mule deer (Odocoileus hemionus hemionus)

Summary Chronic wasting disease (CWD), a progressive neurological disorder of captive mule deer, blacktailed deer, hybrids of mule deer and white-tailed deer and Rocky Mountain elk, is characterized neuropathologically by widespread spongiform change of the neuropil, intracytoplasmic vacuolation in neuronal perikarya and astrocytic hypertrophy and hyperplasia. We report the topographic distribution of amyloid plaques reactive to antibodies prepared against scrapie amyloid in CWD-affected captive mule deer (Odocoileus hemionus hemionus). Scrapie amyloid-immunoreactive plaques were found in the cerebral gray and white matter, in deep subcortical nuclei, in isolation or in clusters in areas of vacuolation, and perivascularly, in subpial and subependymal regions. In the cerebellum, immunoreactive amyloid plaques were observed in the molecular, pyramidal and granular layers. Scrapie amyloid-immunoreactive deposits were also seen in neuronal perikarya. Furthermore, amyloid plaques in CWD-affected captive mule deer were alcianophilic at 0.3 M magnesium chloride indicating the presence of weakly to moderately sulfated glycosaminoglycans. Our data corroborate that CWD in captive mule deer belongs to the subacute virus spongiform encephalopathies.     

Fibrils in brains of Rocky Mountain elk with chronic wasting disease contain scrapie amyloid

Summary Chronic wasting disease (CWD), a progressive, fatal neurological disorder of captive mule deer and Rocky Mountain elk, is characterized neuropathologically by spongiform change in the neuropil, intraneuronal vacuolation and astrocytic hypertrophy and hyperplasia. Recently, scrapie amyloid-immunoreactive plaques have been demontrated in brain tissues of CWD-affected captive mule deer, Rocky Mountain elk and hybrids of captive mule deer and white-tailed deer. We now report on the presence of abnormal fibrils isolated from brain tissues of Rocky Mountain elk using negative-stain electron microscopy. These fibrils resemble those found in scrapie-infected hamster brain. Furthermore, protein bands with relative molecular masses of 26 to 30 kilodaltons were shown to be immunoreactive to antibodies raised against scrapie amyloid by Western immunoblotting. Immuno-dot blot showed similar reactivity. Our data support the clinical and pathological diagnosis of the disease and provide further evidence that CWD belongs to the subacute spongiform encephalopathies.Supported by the Sonderforschungsbereich 194, Deutsche Forschungsgemeinschaft (to D.C.G.)     

Early events in rabies virus infection of the central nervous system in skunks (Mephitis mephitis)

Twenty-four striped skunks were inoculated intramuscularly (long digital extensor muscle of right pelvic limb) with street rabies virus. Groups of two clinically normal skunks were killed at various times after inoculation; skunks that developed rabies were killed in early stages of the clinical signs. Four clinically normal skunks (numbered 1–4) had slight infection in lumbar spinal ganglia, spinal cord and brain. These four skunks were used for detailed immunohistochemical (rabies antigen) studies that included examination of sections from every segment of the spinal cord, most of the spinal ganglia from the 2nd cervical to the 2nd coccygeal (sections at 25-μm intervals of lumbar, sacral and coccygeal ganglia) and brain (sections at 50-μm intervals). In skunks 1–4, there was increasing distribution of antigen-containing neurons that was not correlated with the time elapsed since inoculation. In three skunks (nos. 1, 2 and 3), antigen-containing neurons were predominantly in caudal regions of the spinal cord, caudal right lumbar and sacral spinal ganglia and certain nuclei/regions of the brain (medial reticular formation, right interpositus and lateral vestibular nuclei, left red nucleus, left motor cortex, and left reticular nucleus of the thalamus). Skunk 4 had more extensive infection than skunks 1–3, but the previous pattern was still evident. The results are consistent with viral entrance into the lumbar spinal cord, initial replication mainly at the L2 and L3 levels, local spread in the cord by propriospinal neurons and early transit to the brain via long ascending and descending fiber tracts (bypassing the grey matter of the rostral spinal cord). These mechanisms could provide for early and rapid dissemination in the brain before a significant immune response develops and could induce behavioral changes before the animal is incapacitated by extensive spinal cord infection. Based on the distribution of antigen-containing neurons, the tracts considered most likely to serve as viral transitways from spinal cord to brain include: rubrospinal, corticospinal, spinothalamic, spino-olivary, vestibulospinal and/or spinovestibular, reticulospinal and/or spinoreticular, cerebellospinal and/or spinocerebellar, and dorsal column pathways. Received: 21 April 1995 / Revised: 24 July 1995 / Accepted: 2 August 1995     

Deposition of disease-associated prion protein involves the peripheral nervous system in experimental scrapie

There is some evidence that the peripheral nervous system (PNS) is involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). The TSE-specific abnormal prion protein (PrP^sc) is considered as surrogate marker for infectivity. We traced the deposition of PrP^sc by immunocytochemistry in sheep and hamsters inoculated intraperitoneally with scrapie. The trigeminal, dorsal root, celiac, thoracic, and nodose ganglia contained ganglion cells and fewer satellite cells with prominent granular PrP^sc deposition. As a novel deposition pattern, punctate deposits in adaxonal location were seen along nerve fibers of peripheral nerve adjacent to ganglia. Such prominent involvement of the PNS in two different experimental scrapie models emphasizes the need to consider the PNS in natural scrapie and other TSEs including bovine spongiform encephalopathy as potential source of infectivity. Received: 5 May 1999 / Revised, accepted: 5 July 1999     

Neuronal autophagy in experimental scrapie

Summary In this study we report the formation of giant autophagic vacuoles (AV) in neurons in experimental scrapie in hamsters. Autophagy is an important step in the cellular turnover of proteins and organelles. It is known to occur in neurons under physiological as under pathological conditions. Giant AV, however, are seen very rarely only in pathological states. In our model AV are much more numerous after intracerebral (i.c.) transmission of the scrapie agent than after the transmission via the intraperitoneal route which points to a correlation between the intensity of the process and the period of incubation. As the appearance of the AV in our model is correlated chronologically with that of scrapie-associated fibrils, at least after i.c. transmission, the process may be related to a disturbance of cellular protein metabolism and, thus, to the processing of prion protein.     

Human and experimental spongiform encephalopathies: Recent progress in pathogenesis

The spongiform encephalopathies belong to the group of “slow virus infections” of the nervous system, characterized by a long incubation period, a protracted course and involvement of the nervous system with a lethal outcome. In contrast to the conventional virus infections, such as visna in sheep and progressive multifocal leukoencephalopathy (PML) in humans, the etiological agent for the spongiform encephalopathies has not been clearly defined. The known forms in animals are scrapie in sheep and goats, transmissible mink encephalopathy, and chronic wasting disease of mule deer and elk. In humans, the three known forms are Kuru, now mainly of historical interest, Creutzfeldt-Jakob (CJ) disease and the syndrome of Gerstmann-Straussler-Scheinker (GSS). An important feature of these diseases is the lack of an immune response by the host, which is reflected in the absence of inflammatory infiltrates in the affected tissues. In this editorial the two most important hypotheses on the etiology and pathogenesis of this group of conditions will be discussed. The “prion” hypothesis considers the possibility that a protein, derived from a normal component of the neuronal membranes may have a leading role, not only in the infectivity and transmissibility of these diseases, but in the pathological changes that ensue. A single host gene would code for both the normal and altered proteins. The altered protein would be partially insoluble and would result in the deposition of fibrils and rods which would precipitate in the form of amyloid. Since the involved protein would be coded for by the host, there would be no immune response against it. The viral hypothesis, on the other hand, maintains that the difficulties in isolating a nucleic acid in the infective agent are probably due to technical limitations more than to its real absence. A scrapie-specific nucleic acid, for instance, could be enveloped in a protein encoded by the host, so that the immune system would not be stimulated. Alternatively, a conventional virus, particularly a retrovirus, might be responsible for these diseases, since retroviruses can be integrated into the host genome, thus remaining hidden from the immune system of the host. Data, both in favor and against these two major hypothese will be briefly presented.

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Sommario Le encefalopatie spongiformi fanno parte del gruppo delle “infezioni lente” del sistema nervoso, caratterizzate da un lungo periodo di incubazione, un corso clinico prolungato ed interessamento del sistema nervoso ad esito letale. A differenza delle infezioni da virus convenzionali, quali per esempio “visna” nella pecora e “progressive multifocal leukoencephalopathy” (PML) nell'uomo, l'agente eziologico delle encefalopatie spongiformi non è stato ancora decisamente individuato. Le forme conosciute nel mondo animale sono “scrapie” nella pecora e nella capra, recentemente trasmessa anche a piccoli roditori da laboratorio, “transmissible mink encephalopathy” e “chronic wasting diasease of the mule deer and elk”. Nell'uomo, le tre forme conosciute sono “Kuru”, ormai di valore essenzialmente storico, la malattia di Creutzfeldt-Jakob (CJ) e la sindrome di Gerstmann-Straussler-Scheinker (GSS). Una caratteristica importante di queste malattie è la mancanza di una risposta immunitaria da parte dell'ospite, la quale si traduce in assenza di infiltrati infiammatori nei tessuti nervosi affetti. In questo editoriale, le due ipotesi più importanti sulla eziologia e patogenesi di questo gruppo di malattie verranno discusse. L'ipotesi “prion” considera la possibilità che una proteina, derivante da una normale componente delle membrane neuronali, possa avere un ruolo principale sia nella infettività e trasmissibilità della malattia che nel risultante processo patologico. Un unico gene dell'ospite potrebbe essere il codice sia per le normali che per le alterate proteine. La proteina alterata sarebbe parzialmente insolubile e darebbe luogo a deposizione di fibrille che precipiterebbero in forma amiloide. Poiché la proteina interessata è codificata dall'ospite, non ci sarebbe risposta immune contro di essa. L'ipotesi virale, d'altronde, sostiene che le difficoltà di isolare un acido nucleico nell'agente infettivo è probabilmente dovuta più a limitazioni tecniche che ad una sua effettiva mancanza. Un acido nucleico specifico per la scrapie, per esempio, potrebbe essere completamente circondato da un proteina codificata dall'ospite, in maniera che il sistema immunitario non verrebbe a contatto con esso. Oppure, un vero virus convenzionale, quale un piccolo retrovirus, potrebbe essere responsabile della malattia, dato che i retrovirus hanno la capacità di integrarsi nel genoma dell'ospite, rimanendo, di nuovo, invisibili alle cellule immunitarie. Dati, sia in favore, che contro queste ipotesi opposte, verranno brevemente presentati.

     

Focal and asymmetrical vacuolar lesions in the brains of mice infected with certain strains of scrapie

Summary In mice experimentally infected with most strains of scrapie, vacuolar degeneration almost always shows a bilaterally symmetrical distribution in the brain. However, asymmetrical foci of vacuolation are frequently seen with a group of six mouse-passaged isolates from diverse natural sheep sources (designated 31A, 51C, 87A, 125A, 138A and 153A). As these isolates are similar in other respects they may be independent isolations of the same strain of scrapie. The distribution of focal vacuolar lesions in C57BL mice affected with 87A scrapie was found to depend on route of infection. In mice injected intracerebrally into the left or right hemisphere, all focal asymmetrical lesions occurred on the side of injection, in some cases intense vacuolation being associated with the needle scar. following midline intracerebral injection, focal lesions were evenly distributed between the two sides and were most frequent in the medial areas of the thalamus. In one mouse injected intraocularly, intense unilateral lesions were seen contralaterally in the major retinal projection regions. Asymmetrical lesions also occurred following infection by intraperitoneal, intravenous and subcutaneous routes, but were less frequent than after intracerebral infection. The most likely explanation is that focal asymmetrical lesions result from focal replication of scrapie infectivity in the brain. As all the scrapie strains which frequently produce asymmetrical vacuolation are also those that generate mutants, it is possible that focal lesions represent foci of the new mutant strain, replicating preferentially in areas with a low background level of the parent strain.     

The long incubation period in rabies: delayed progression of infection in muscle at the site of exposure

The striped skunk (Mephitis mephitis) is a host of rabies in large areas of Canada and the United States. In each of two experiments, equal numbers of skunks in two groups were inoculated intramuscularly with low doses of a field strain of rabies virus (street rabies virus). In each experiment, skunks in one group surviving to 2 months were killed at this time and selected tissues were used for examination by the polymerase chain reaction (PCR) method or by immunohistochemistry for rabies antigen. Results of detailed examinations using PCR technology (experiment 1) indicated that muscle at the inoculation site contained viral RNA at 2 months postinoculation, when other relevant tissues on the route of viral migration and early entrance into the central nervous system were negative. The cellular location of virus/antigen, as determined immunohistochemically in experiment 2, was striated muscle fibers and fibrocytes. Our results indicate a major role of muscle (tissue) infection at the inoculation site in the long incubation period of rabies in skunks. These and related findings will be useful in rabies control and, if applicable to other species, will be relevant in postexposure treatment. Received: 31 July 1996 / Revised, accepted: 11 November 1996     

Altered plasma membranes in experimental scrapie

Summary The status spongiosus in the cerebral cortex of mice affected with two different strains of scrapie virus corresponded to focally swollen perikaryal cytoplasm of nerve cells and astrocytes, to swollen neuronal and astrocytic processes and to membrane-bounded vacuoles within pre- and postsynaptic neuronal terminals. The swollen cytoplasm contained uniformly dispersed, finely granulo-filamentous material. A few enlarged dendrites were filled with fragments of membranes or 350 Å wide vesicular and tubular structures suggestive of virus particles. Ruptured plasma membranes and curled fragments of membranes were seen around cleared cytoplasmic regions and within membrane-bounded vacuoles. Neurons or astrocytes that lined affected cells or processes frequently showed similar changes. Confluence of swollen cells or processes occurred after dissolution of their adjacent plasma membranes. Astrocytes reacted to the injury by proliferation whereas nerve cells degenerated. The findings are compared to those seen in other subacute spongiform virus encephalopathies, i. e., mink encephalopathy, Kuru and Creutzfeldt-Jakob disease. The characteristic vacuolar degeneration of nerve cells in these diseases which is associated with fragmentation and accumulation of plasma membranes is discussed with reference to the peculiar properties of the scrapie virus.This investigation was supported in part by United States Public Health Research Grant NS-09053 from the National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland.     

Neuronal autophagic vacuoles in experimental scrapie and Creutzfeldt-Jakob disease

Summary We report the presence of autophagic vacuoles (AV) in neuronal perikarya and neuronal processes of rodents with experimental scrapie and Creutzfeldt-Jakob disease. AV were composed of sequestrated cytoplasmic areas containing ribosomes and occasionally mitochondria and small secondary vacuoles. The formation of AV may contribute to neuronal degeneration and ultimately to neuronal loss.P. P. Liberski is a recipient of a fellowship from the Fogarty International Center and a grant from the Polish Academy of Sciences (VIII/40)     

Apoptosis in experimental rabies in bax-deficient mice

The challenge virus standard (CVS) of fixed rabies virus produces a fatal encephalitis in adult and suckling mice after intracerebral inoculation. The infection is associated with apoptotic cell death in brain neurons and increased immunoreactivity to the Bax protein in the hippocampus and cerebral cortex. Five- to 7-day-old bax-deficient mice and their wild-type littermates were inoculated intracerebrally with either CVS or the RV194-2 variant of rabies virus, which is avirulent in adult mice after intracerebral inoculation. The clinical disease was similar with both viruses in bax-deficient and wild-type mice with 100% mortality. CVS produced similar apoptotic changes in bax-deficient and wild-type mice, except that apoptosis was more marked in neurons of the dentate gyrus and cortical neurons in the wild-type mice. After inoculation with RV194-2, the morphologic changes of apoptosis were markedly less severe in the cerebral cortex, hippocampus, and cerebellum of the bax-deficient mice than wild-type mice. However, apoptotic changes were moderate to severe in the brain stem in both wild-type and bax-deficient mice with both viruses. Although apoptotic cell death was much less prominent in bax-deficient mice after inoculation with RV194-2, apoptosis of infected brain stem neurons occurred in this fatal infection. Although the Bax protein plays an important role in modulating rabies virus-induced apoptosis under specific experimental conditions, other modulators are also likely important. Received: 23 November 1998 / Revised, accepted: 27 January 1999     

Ultrastructural evidence that ependymal cells are infected in experimental scrapie

During the last stage of infection in the experimental scrapie-infected hamster model, light microscopy reveals typical immunostaining of PrPsc in the subependymal region and at the apical ependymal cell borders. Whereas the subependymal immuno-staining is known to originate from extracellular amyloid filaments and residual membranes of astrocytes as constituents of plaque-like structures, the ultrastructural correlate of the supraependymal PrPsc staining remains uncertain. To decipher this apical PrPsc immunopositivity and subsequently the ependymocyte-scrapie agent interaction, we employed highly sensitive immuno-electron microscopy for detecting PrPsc in 263K scrapie-infected hamster brains. The results revealed the supraependymal PrPsc signal to be correlated not only with extracellular accumulation of amyloid filaments, but also with three distinct ependymal cell structures: (1) morphologically intact or altered microvilli associated with filaments, (2) the ependymal cell cytoplasm in proximity of apical cell membrane, and (3) intracytoplasmic organelles such as endosomes and lysosomal-like structures. These findings suggest a strong ependymotrope feature of the scrapie agent and recapitulate several aspects of the cell-prion interaction leading to the formation and production of PrPsc amyloid filaments. Our data demonstrate that in addition to neurons and astrocytes, ependymocytes constitute a new cellular target for the scrapie agent. In contrast, the absence of PrPsc labeling in choroid plexus and brain vascular endothelial cells indicates that these cells are not susceptible to the infection and may inhibit passage of the infectious agent across the blood-brain barrier.     

Neonatal status convulsivus, spongiform encephalopathy, and low activity of Na+/K(+)-ATPase in the brain.

The first and second child of a family died from neonatal seizures with no detectable brain malformation, metabolic, infectious, or chromosomal etiology. Neuropathological examination of the brain of the second child who died at 11 days revealed a widespread spongy state and a selective vulnerability of the astrocytes characterized by numerous enlarged bare astrocytic nuclei and different forms of astrocyte degeneration. The glial cells were strongly positive for glial fibrillary acidic protein and vimentin immunocytochemical reaction. Cortical measurement of Na+/K(+)-ATPase revealed very low enzyme activity. We hypothesize that a defect of Na+/K(+)-ATPase of the astrocytes could be the common pathogenetic factor for the congenital status convulsivus and for the spongy state.     

Retinal damage in scrapie mice

Summary Retinal damages in mice infected with serapie are reported. The effects ranged from no histopathological changes, through partial loss of the outer nuclear layer (ONL) to the most severe changes with complete loss of ONL cells and photoreceptors. For the most part, cells of the inner nuclear and ganglion layers were spared. These changes were found in 23% of C57BL/6J mice injected with the ME7 strain of scrapie and in 28.5% of VM mice injected with the 87V strain, while no changes were found in 13 IM mice injected with the 87V strain of scrapie. The possible relation of these changes to scrapie infection and to light induced retinopathy is discussed.     

Tubulovesicular particles occur early in the incubation period of murine scrapie

Tubulovesicular bodies are structures, apparently specific to the transmissible spongiform encephalopathies, which are of unknown composition and significance. Prion protein (PrP) is absent from tubulovesicular bodies when tissues are examined by immunogold electron microscopy. In the F1 cross of C57 and VM mice (CVF1) infected with ME7 scrapie there is a marked degeneration of hippocampal CA1 neurons. In this model the earliest changes seen, at about 100 days post inoculation (dpi) are a degeneration of axon terminals and synaptic loss. Terminal disease is around 250 dpi. In blind coded trials we counted the number of tubulovesicular particles and estimated their density in 56–76 electron micrographs taken from the stratum radiatum of each of one or two CVF1 ME7-infected mice at 84, 100, 126, 154 and 181 dpi and from four normal brain inoculated control mice. Tubulovesicular particles were present from 98 dpi and the density of particles increased with increasing incubation period. The very early occurrence of tubulovesicular particles, before the presence of significant pathology, argues that tubulovesicular particles are a part of the primary disease and are not epiphenomena. Received: 28 June 1999 / Revised: 30 August 1999 / Accepted: 6 September 1999     

Apoptotic cell death in experimental rabies in suckling mice

A fatal encephalomyelitis developed after intracerebral inoculation of 6-day-old ICR mice with the challenge virus standard (CVS) strain of fixed rabies virus. The brains of CVS-infected mice showed widespread morphologic changes of apoptosis, which were particularly prominent in pyramidal neurons of the hippocampus and in the cerebral cortex. Evidence of oligonucleosomal DNA fragmentation was sought in situ using the TUNEL method. TUNEL staining was observed in many neurons, and rabies virus antigen was usually demonstrated with immunoperoxidase staining in similar regions. Neurons in the dentate gyrus of the hippocampus demonstrated expression of viral antigen, apoptotic changes, and positive TUNEL staining. This region normally demonstrates little infection in CVS-infected adult mice. Double labeling of neurons with TUNEL and viral antigen indicated that infected neurons actually underwent apoptosis. Increased immunoreactivity against the Bax protein was demonstrated compared to uninfected mice. Purkinje cells expressed viral antigen, but did not show significant morphologic changes of apoptosis or TUNEL staining. In contrast, neurons in the external granular layer of the cerebellum did not express viral antigen, but demonstrated greater morphologic changes of apoptosis and positive TUNEL staining than uninfected controls. Apoptotic cell death likely plays an important role in the pathogenesis of rabies virus infection in suckling mice. There was evidence of more apoptosis in the brains of suckling mice than in those of adult mice and this finding explains the greater neurovirulence of rabies virus in younger mice. Rabies virus likely induces apoptosis in vivo by both direct and indirect mechanisms. Received: 15 July 1997 / Revised: 25 August 1997 / Accepted: 8 September 1997     

Electrophysiological and sleep alterations in experimental mouse rabies

Changes in the spontaneous brain electrical activity and sleep organization were investigated in 5 mice strains during the evolution of experimental fixed rabies infection. Cortical electrodes were chronically implanted for continuous EEG recording and spectral analysis until death. Three evolutionary phases were individualized. The initial phase exhibited alterations of sleep stages, REM sleep disappearance, pseudoperiodic facial myoclonus and first clinical signs. The mature phase was characterized by a generalized EEG slowing (2-4 cycles/s). The terminal phase occurring with extinction of hippocampal rhythmic slow activity showed a flattening of cortical activity. The brain electrical activity ceased about 30 min before the cardiac arrest. Paroxysmal activities appeared during the course of the disease as bursts of rhythmic slow waves, pseudoperiodic spikes or occasionally ictal epileptic discharges. Spectral analysis revealed a progressive and characteristic clustering of the EEG frequency band power values. The spread of infection in the CNS was monitored by specific immunofluorescence studies which revealed the presence of rabies virus antigen in the pons, the cerebellum, the thalamus and the cortex during the initial phase. The pyramidal field of the hippocampus was infected during the mature phase but the gyrus dentatus was never infected even at the terminal phase. These studies show that particular neuronal functions are impaired during rabies virus infection suggesting that neuronal alterations may be involved in the pathogenic mechanisms leading to lethality.     

Appearance of tubulovesicular structures in experimental Creutzfeldt-Jakob disease and scrapie preceeds the onset of clinical disease

Summary We have consistently observed tubulovesicular structures in brain tissues during the terminal stages of naturally occurring and experimentally induced spongiform encephalopathies, irrespective of the host species and virus strain. In NIH Swiss mice inoculated intracerebrally or intraocularly with the Fujisaki strain of Creutzfeldt-Jakob disease (CJD) virus, tubulovesicular structures, measuring 20–50 nm in diameter, were particularly prominent in dilated, pre-and postsynaptic neuronal processes, occasionally being mixed with synaptic vesicles. These structures appeared 13 weeks following intracerebral inoculation, 5 weeks before the onset of clinical signs, when spongiform changes were also detected. The number and density of tubulovesicular structures increased steadily during the course of clinical disease, and were particularly abundant in mice 47 to 51 weeks after intraocular inoculation. In hamsters infected with the 263 K strain of scrapie virus, these structures were initially detected 3 weeks following intracerebral inoculation and increased dramatically at 10 weeks postinoculation. The appearance of tubulovesicular structures before the onset of overt disease in mice inoculated with CJD virus by either the intracerebral or intraocular route, and before the appearance of other neuropathological changes in hamsters infected with scrapie virus, indicate that they represent either a part or aggregate of the infectious virus or a pathological product of the infection.Presented in part at the 64th annual meeting of the American Association of Neuropathologists, held in Charleston, South Carolina, June 9–12, 1988 and at the 7th annual meeting of the American Society for Virology, Austin, Texas, June 12–16, 1988. Dr. Pawel P. Liberski is a recipient of a fellowship from the Fogarty International Center and a grant from the Ministry of Health and Social Welfare, Poland