Epi/op.\io, 19257-264, 1978 Raven Press, New York

Appearance of EEG Changes Reminiscent of a Secondary Generalized Epilepsy in a Rhesus Monkey Inoculated with a Strain of Kuru :::J. Bert, t G . Vuillon-Cacciuttolo, tE. B a l z a m o , 3P. De Micco, OD. Gambarelli, $J. T a m a l e t , and '!'H. G a s t a u t

In 1973, Grabow et al. succeeded i n transmitting mink encephalopathy to the squirrel monkey and observed generalized or focal convulsive seizures and myoclonia in several monkeys. The same authors. however, also transmitted to the squirrel monkey a human spongiform encephalopathy of uncertain diagnosis, which produced neither myoclonic nor convulsive seizures in the animals (Grabow et al., 1976). Finally, A s h e r (1975) o b s e r v e d only abnormal movements with no epileptic character in monkeys inoculated with a strain of tick-borne encephalitis virus w h i c h , in m a n , is capable of causing Kojevnikov type epilepsia. Electroencephalographic data are rarer but no less varied. In para-influenza virus disease of the chimpanzee accompanied by clinical epileptic seizure, Lief (1976) d e s c r i b e s slow abnormalities with n o specific character. Similarly, in experimental kuru of the chimpanzee. Niedermeyer et al. (1972) and Asher et al. (1973) observed only slow abnormalities without any epileptic character. However, in exReceived November 17, 1977. perimental Creutzfeld - Jakob disease of Key words: EEG-Slon, virrcJ ~ i ; ~ ~ , ( r s t , , s - K i i r i ~ LtJt7fl(J.Y - G(l,ytclut s\ildrotn ~~-Rht'sltS in O t l k ? . . the chimpanzee, Cathala et al. (1974) and

Various types of epileptic phenomena are frequently but not systematically observed in experimental "slow virus" diseases of the central nervous system. T h u s , Lief et al. (1976) elicited epilepsy with episodic generalized seizures in 2 chimpanzees inoculated at birth with a para-influenza virus strain; myoclonic and convulsive seizures are common in experimental CreutzfeldJakob disease in all successfully inoculated primate species (Gibbs and Gajdusek, 1969; Gibbs and Gajdusek, 1971; Cathala et al., 1974; C o u r t e t a l . , 1975; Gibbs and Gajdusek, 1976). It should be emphasized, however, that the epileptic manifestations of experimental Creutzfeld- Jakob disease are not constant (in 2 M a c . ~ c aurctoi'dcs inoculated with the same Creutzfeld- Jakob s t r a i n , E s p a n a e t a l . (1975) o b s e r v e d myoclonic phenomena in only one of the animals) and that myoclonic and convulsive seizures are very rare in experimental kuru in the chimpanzee ( 2 animals out of 41, and only in the terminal stage of the disease (Asher et al., 1973)).

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Court et al. (1975) observed the early appearance of generalized spike-and-wave discharges and subsequently the development of periodic triphasic waves. And in mink encephalopathy of the squirrel monkey, Grabow et al. (1973) observed epileptiform activities in several monkeys and periodic complexes in 2 animals. The same authors (Grabow et al., 1976) also observed periodic di- or triphasic complexes in 1 of several squirrel monkeys inoculated with human spongiform encephalopathy . The available EEG studies clearly show that the E E G abnormalities observed in these experimental encephalopathieswhether the diseases involve conventional viruses or unconventional agents-are distinctly differentiated from the characteristic abnormalities observed in primary or secondary generalized human epilepsies: spike-and-wave complexes and especially fast or slow spike-and-wave complexes, either bisynchronous o r diffuse in both hemispheres. Except for Grabow’s observations involving mink encephalopathy of the squirrel monkey and Cathala and Court’s observations of Creutzfeld-Jakob disease of the chimpanzee, these reports describe slow abnormalities without any specific character, with the appearance in several animals of periodic complexes suggesting the EEG abnormalities frequently encountered in Creutzfeld -Jakob disease. For this reason it seems appropriate here to present observation data concerning 1 of 10 rhesus monkeys inoculated with a kuru strain passaged on a rhesus monkey (Enage strain, rhesus L6 56) in Dr. Gajdusek’s laboratory. Two animals from this group were sacrificed 11 and 16 months after inoculation in order to study precocious lesions. Two others, inoculated in May and June 1975, have presented no symptoms and remain under observation. The 6 remaining monkeys developed an experimental disease with variable numbers of myoclonic jerks and major irritative signs on the EEG tracings (the clinical and E E G

Epilepsia, Vol. 19, June 1978

data obtained during this study are currently being published). During the 4 month mature phase of the disease in 1 of the monkeys (Mm 07), the animal presented EEG abnormalities very similar if not identical to those observed in secondary generalized epilepsies and more particularly in Lennox-Gastaut syndrome. This syndrome, peculiar to children, presents a characteristic semiological triad: ( I ) epileptic seizures of various types, of which the most evocative are-apart from atypical absences-minor motor seizures, that is to say, massive bilateral myoclonia, tonic seizures (mostly nocturnal, occasionally repeated in true daily nocturnal status epilepticus), and atonic attacks: ( 2 ) mental retardation and occasional deterioration; (3) E E G alterations taking the form of slower background rhythms and epileptic paroxysms, whether interictal (diffuse slow spike-and-wave complexes repeated pseudorhythmically at approximately 2 cycles/ sec and/or focalized and often multiple spike bursts) or ictal (essentially involving desynchronization of the background rhythms or rapid synchronization-recruiting epileptic rhythm-during the minor motor seizures). This rhesus monkey, like all the others in the group, was inoculated in the left frontal lobe transorbitally (0.2 ml of dilution of a 20% cerebral solution in P.B.S., pH 7.4) and intravenously (0.2 ml of cerebral suspension) on May 14, 1975. Prior to inoculation, electrodes were surgically implanted on the surface of the dura mater for E E G recording ( 1 1 electrodes) and around the perimeter of the orbits to record eye movements (4 electrodes). Several control tracings were recorded prior to inoculation. Tracings were subsequently obtained in a 12 hr nocturnal recording session (6:OO p.m. to 6:OO a.m.) every 4 months, then at monthly intervals for the 18th, 19th, and 20th months, and finally every 2 weeks. The first characteristic EEG abnormalities appeared in the waking state during

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FIG. 3. The tracing shown in the preceding figure is characterized by the occurrence of very numerous tonic seizures, one of which is taken as an example. Prior to the seizure the complex background activity is characterized by a predominant 6 cyclesisec theta rhythm. On the penocular channel (considerably perturbed by the frontal EEG) the seizure is shown by interferential EMG activity corresponding to tonic contraction of the facial muscles. On the EEG it is shown by a 6 sec desynchronization of the background activity, followed by a 10 sec pseudorhythmical burst of diffuse slow spike-and-wave complexes before resumption of normal background activity.

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EEG CHANGES IN O N E KURU-INOCULATED MONKEY

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were replaced by highly degraded slow spike-and-wave complexes. From a clinical standpoint, a true dementia syndrome rapidly developed from the 20th month with continuous deambulation but with no cerebellar signs. A few spontaneous myoclonic j e r k s were observed, but not generalized or focal convulsive seizures. This monkey was sacrificed on June 15, 1977. N o cytopathic o r hemagglutinant effects were revealed by inoculating a cerebral suspension from the animal to Vero, BSCI, KB, Hela cell cultures after three blind passages on these cells and after co-culture of brain cells from this animal with primoexplanted simian cells in the presence of lysolecithin. The serum of this animal contained no antibodies capable of complement neutralization and fixation for paramyxoviruses, enteroviruses, o r herpes viruses. Microscopic examination revealed a widespread status spongiosus accompanied by vacuolation, degeneration, loss of

nerve cells, and gliosis. These alterations extended widely through the cerebral cortex, neostratum, and thalamus. The dentate nucleus of the cerebellum and the pontine nuclei were less involved (Fig. 5). Aseptic samples of the cerebral cortex taken just before the animal’s death were used to inoculate 4 new rhesus monkeys on July 26, 1977. Thus, for 3 months this monkey presented EEG abnormalities comparable to those observed in generalized epilepsies, first in primary generalized (i.e., “functional”) epilepsies, then, under the effect of a degenerative process, in secondary generalized epilepsies and particularly in Lennox-Gastaut syndrome. Electroencephalographic criteria clearly distinguish this monkey from the other 5. All the animals presented more or less pronounced irritative EEG signs, and 4 of them presented electroencephalographically and clinically typical “tonic seizures” during sleep; only Mm 07, however, presented all Epilepsia, Vol. 19, June 1978

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FIG. 5 . Cortical lesions: Left: status spongiosus associated with loss of neurons and astrocyte proliferation in the insular cortex ( ~ 2 2 0 )Right: ; coarse intracytoplasmic vacuolation of neuron in the cingulate gyrus ( ~ 4 0 0 ) .

the characteristics of this form of epilepsy. To the authors’ knowledge, this is the first time that the electrographic symptoms of Lennox -Gastaut syndrome have been reproduced experimentally. Obviously no effort is made here to suggest that this syndrome can be elicited by an unconventional virus. Three major differences distinguish this experimental disease from Lennox- Gastaut syndrome: 1 . The neuropathological data are completely different. The brain of monkey Mm 07 presented very extensive status spongiosus lesions which do not exist in LennoxGastaut syndrome. Nevertheless, it should be noted that this difference is perhaps less fundamental than it might first appear. The significance of the status spongiosus, which is always extensive in experimental kuru and Creutzfeld-Jakob disease, is unclear (Fraser and Dickinson, 1973). Creutzfeld-Jakob disease in man preEpilepsfa, Vol. 19, June 1978

sents major cortical status spongiosus, but the EEG abnormalities characterizing this disease (periodic di- o r triphasic complexes) are of a different type from those observed in this rhesus monkey. Scrapie, moreover, is an animal disease caused by a virus of similar nature to that of kuru or Creutzfeld-Jakob disease, and yet in the natural disease in several species of sheep the status spongiosus lesions may be absent (Dickinson, 1976). 2 . The clinical troubles presented by monkey Mm 07 bear only a slight resemblance to the clinical signs of LennoxGastaut syndrome, in which the very frequent nocturnal tonic seizures are generally associated with diurnal myoclonic seizures. tonic seizures, and atonic seizures. 3. After an 18 month silent incubation period, the disease evolved in 6 months to a fatal outcome as with all experimental spongiform encephalopathies. This course

EEG CHANGES IN O N E KURU-INOCULATED MONKEY bears no resemblance to the chronic extended evolution of Lennox-Gastaut syndrome. With these distinctions clearly established, this observation is of considerable interest, since it shows that an unconventional virus, which virtually never elicits epileptic clinical or EEG signs in man or in the chimpanzee, is capable of inducing an epileptogenic encephalopathy in another primate species. Furthermore, it must be stressed that this encephalopathy presents a number of electrographic characteristics which identify a peculiar form of human epilepsy: a very specific EEG syndrome is thus reproduced. Of the 6 rhesus monkeys which developed an experimental disease, only 1 presented this set of EEG signs. These two aspects-the particularity of experimental kuru in the rhesus monkey and the singular EEG character of the disease in monkey Mm 07-emphasize the importance of genetic and individual factors in determining the characteristics of encephalopathies caused by unconventional viruses. This suggests that infectious agents with no epileptogenic potential in laboratory animals could induce epileptogenic encephalopathies in man. For several years, one of the authors (H.G.) has advanced the hypothesis that certain cases of Lennox-Gastaut syndrome might be of viral origin. The observation discussed here does not provide a supporting argument for this hypothesis, but it does show t h a t an unconventional virus may cause an experimental disease accompanied by the abnormal EEG patterns which characterize this syndrome. It shows also that the development of this experimental syndrome is related not only to the infectious agent but to the individual characteristics of the host as well. Such considerations clearly underscore the pathogenic complexity of these diseases and explain the difficulties encountered in experimental reproduction. They also designate an un-

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doubtedly promising research field opened up by the fundamental pioneering work of Gajdusek and Gibbs.

SUMMARY In a group of rhesus monkeys (Mtrctrctr in 11 la t t ( I ) in o c u 1ate d in t ra c e re b ra I I y and intravenously with a strain (Enage strain rhesus L6 56) of kuru already passaged in rhesus monkeys, 1 monkey presented the typical EEG pattern of epileptogenic encephalopathy reminiscent of the LennoxGastaut syndrome. This observation provides no direct evidence for the viral origin of epilepsies of this type. It does, however, show that it is possible to induce an epileptogenic encephalopathy by an unconventional infectious agent.

ACKNOWLEDGMENTS Our thanks are due to Doctors FranGoise Cathala, Carleton Gajdusek, and Clarence Gibbs for advice and profitable discussions. They kindly provided us with the kuru virus strain. This work was partly supported by a grant of the French Foundation for Medical Research.

REFERENCES Asher DM. Movement disorders in rhesus monkeys after infection with tick-borne encephalitis virus. In: BS Meldrum and C D Marsden (Eds). Prit?z(r/eA4otlcl.s of’Ncr~rolopictrl LXsortlc~..\.V o l 10: Ad\~rt~c~c,.t it7 N r i r r o l o g ~ .Raven Press. New York. 1975. uu 277-289. Asher D M , Gibbs C J . David E , Alpers MP. and Gajdusek DC. Experimental kuru in the chimpanzee. Symp lVth Int Congr Primat.. Vol. 4: Notr H i o n t i t i f r i t ? i t i r r ~ ( i t i d H i i t i i t i t i L X \ c , t i . s c ~ . ~ . Karger. Basel. 1973, pp 43-9U. Cathala F. Court L, Rohmer F. Gajdusek DC. Gibbs CJ, and Castaigne P. Experimental transmission of Creutzfeld-Jakob disease to chimpanzee. with an electroencephalographic study using indwelling electrodes. In: A Subirana and JM Burrows (Eds).

x

Proc~l~c~ditl,~.s o f 1/11, Itltl’r.lltrtiotltrI c(~tl,qrl,.\.\of’ Ncr(ro1ogy. Excerpta Medica. Amsterdam, 1974,

pp 381 -389. Court L. Cathala F. Gajdusek DC. and Rohmer F. Modifications du comportement. de l a vigilance et des activites electriques cerebrales au cours d‘une maladie de Creutzfeld-Jakob experimentale d u chimpanze. Rci. EEG Ncirrophy.rio/ 5 : 3 3 5 - 343. 1975.

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Dickinson AG. Scrapie in sheep and goats. In: RH Kimberlin (Ed), Sloira Virus Disc.ases of Animuls mid Man, North Holland, Amsterdam, 1976, pp 209-24 I . Espana C , Gajdusek DC, Gibbs CJ, Osburn BI, Gribble DH, and Cardinet GH. Transmission of Creutzfeld- J a k o b disease t o t h e stumptail macaque (Macac.a urctozdes). Proc Soc Exp B i d M e d 149:723-724, 1975. Fraser H and Dickinson AG. Scrapie in mice. J Comp Path01 83:29-39, 1973. Gibbs CJ, and Gajdusek DC. Infection as the etiology of spongiform encephalopathy (Creutzfeld- Jakob disease). Science 165: 1023- 1025, 1969. Gibbs CJ and Gajdusek DC. Transmission and characterization of the agents of spongiform virus encephalopathies: Kuru, Creutzfeld-Jakob disease, scrapie and mink encephalopathy. In: L P Rowland (Ed), Immunologic~aI Disorders of the Nervous S y s t e m , Vol 4 9 . R e s Pub1 A s s o c R r s Ncrv Ment Dis, Williams & Wilkins, Baltimore, 1971, pp 383-410. Gibbs CJ and Gajdusek DC. Studies on the virus of subacute spongiform encephalopathies using primates, their only available indicators. First Inter-American Conference on Conservation and Utilization of American Non-Human Primates in Biomedical Research. PAHO Scientific Publication no. 317, 1976, pp 83-109. Grabow JD, Zurhein G, Eckroade RJ, Zollman PE, and Hanson RP. Transmissible mink encephalop. athy agent in squirrel monkeys. Neurology 23: 820-832, 1973. Grabow JD, Campbell RJ, Okazaki H , Schut L , Zollman PE, and Kurland LT. A transmissible subacute spongiform encephalopathy in a visitor to the Eastern Highlands of New Guinea. Brain 99:637-658, 1976. Lief FS, Rorke LB, Kalter SS, Hoffman SF, Roosa RA, Moore GT, Cummins LB, McCullough B, Rodriguez AR, Eichberg S, and Koprowski H . Infection and disease induced in chimpanzees with 6/94, a para-influenza type I virus isolated from human multiple sclerosis brain. J Neuropathol Exp Nt,urol 35:644-663, 1976. Niedermeyer E , Gibbs CJ, and Marsh R. EEG studies in subacute and degenerative neurological diseases experimentally produced in ferrets and chimpanzee. Electroencephalogr Clin Neurophysiol 33: 351-352, 1972.

€pi/epsia, Vol. 19, June 1978

RESUME Parmi u n groupe de macaques (Macaca mularra) inoculCs intrackrkbralement et intraveineusement avec une souche de kuru (Enage strain rhesus L6 S6) provenant elle-m&med’autres macaques inoculks, I’un d’eux a prksente des manifestations EEGraphiques evocatrices d’un syndrome de Lennox-Gastaut. Une telle observation ne constitue pas une preuve directe de I’origine virale des Cpilepsies humaines de ce type. Toutefois elle dkmontre qu’il est possible de produire chez certains singes une enckphalopathie kpileptogkne sous I’effet d’un agent infectieux nonconventionnel. (J .-L. Gastaut, Marseilles)

RESUMEN Un mono, de u n grupo de rhesus (Macucu mulatta) inoculados intracerebral e intravenosamente con una cepa (cepa Enage rhesus L6 56) de kuru previamente pasada por monos rhesus, present6 el tipico patr6n electroencefalogrhfico de una encefalopatia epileptogenica reminiscente del sindrome de Lennox-Gastaut. Esta observaci6n no proporciona evidencia directa con respecto a1 origen viral de este tipo de epilepsias. Sin embargo, si demuestra que es posible inducir una encefalopatia epileptogknica mediante u n agente infeccioso no convencional. (A. Portera Sanchez, Madrid)

ZUSAMMENFASSUNG Eine Gruppe von Rhesusaffen ( M a c a c a m u l a t t a ) wurde intracerebral und intravends mit einem Kurustamm infiziert, der schon eine Rhesuspassage durchgemacht hat (Enage strain rhesus L6 56); ein Affe entwickelte das typische EEG-Bild einer epileptogenen Encephalopathie, das an das Lennox-Gastaut Syndrom erinnerte. Diese Beobachtung erlaubt keinen direkten Hinweis auf die virale Ursache der Epilepsien dieser Art. Sie zeigt jedoch, daB es moglich ist, eine epileptogene Encephalopathie durch einen unkonventionellen infektiosen Erreger hervorzurufen. (D. Scheffner, Hc>idelberg)