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Transcript
Slow Virus Diseases of the Central Nervous System D. CARLETON GAJDUSEK, National Institute of Neurological M.D. Diseases and Stroke, National Bethesda, Maryland I will focus more on subacute and chronic degenerative diseases of man than on animal diseases, and will discuss a few slow infections caused by persistent viruses in man wherein the etiology has now been proved. The degree of involvement of the immune mechanism in the pathogenesis of these diseases can be assessed where we now know that a latent, long-lasting virus is present, especially for those diseases in which transmission to an experimental animal is possible and the animal models may be used in the study of pathogenesis. We were not surprised to see, during the past two decades of virology, a long list of viruses which may be present in an animal for months, even years, after first contact with the agent. But when we asked how many viruses had been isolated from a human subject for a second time, three to six months or a year after original isolation, the list of such viruses was very small. At the time we held the first Symposium on Slow, Latent, and Temperate Virus Infections at the National Institutes of Health six years ago,10 the question that faced us was what viruses or other infectious agents came to mind when a virus diagnostic laboratory was confronted with specimens from a subacute, chronic, or recurrent disease. We knew that herpes simplex was found to be latent in man in the dawn of virology. Indeed, most of us here are carrying that virus in our buccal muIN THIS PRESENTATION Received May 17, 1971. Reprints of this entire Research Symposium are available from the ASCP Secretariat, 2100 West Harrison Street, Chicago, Illinois 60612. Institutes of Health, cosal membranes and a wide range of chemical and physical insults can provoke the masked agent to become pathogenic. What other agents were known five to 10 years ago to fall into an eclipse, hidden, or masked phase from which they could be resurrected? Warts may last for years in some children. Lesions of molluscum contagiosum may persist for many months. Rabies may develop in man after incubation periods of well over a year, during which time the virus is slowly spreading from cell to cell in Schwann cells surrounding nerve fibers, as Johnson has demonstrated by immunofluorescent staining technics.16 Moreover, old epidemiologic evidence supported a relationship between herpes zoster and varicella. Subsequently, isolation of the viruses responsible for the two diseases showed them to be indistinguishable. However, some cases of herpes zoster develop within a few days of injury to the same dermatome in which the lesions appear, and this has been interpreted as strong evidence for the latency of varicella virus in dorsal root ganglia cells and its activation by nerve injury. At the time of our 1964 symposium on slow virus infections,10 persistent infection in infants with cytomegalovirus and rubella virus had just been recognized. After intrauterine or neonatal infection with rubella an infant may harbor and shed rubella virus in the respiratory tract for months and perhaps for more than a year. Persistence of the cytomegalovirus in the urine for similarly long periods after con- 320 September 1971 SLOW VIRUS DISEASES O F T H E CNS genital infection was also found. Some infants will react to such persistent infections with cumulative pathology, whereas others show very few, if any, pathologic lesions. Infectious hepatitis, with its recurrences and the obvious transmission of the virus to recipients from blood and blood products of healthy carriers, which came to medical attention in World War II, is another example of persistent virus infection that has been known for decades. Thus, in 1964 the above-mentioned list of persistent agents in man was nearly exhaustive, unless we wanted still to refer to the larger organisms of lymphogranuloma venerium and trachoma as viruses. Since 1964, however, we have been able to add a large number of other agents to the list. I will concentrate my remarks on the four slow and persistent diseases of the CNS in which we have been most thoroughly involved in our laboratory and mention what may be the possibility that, in man, an autoimmune mechanism is involved with the pathogenesis of these slow infections. I do not think there are any "slow" viruses. There may be slow virologists, but not slow viruses. We usually refer to "slow virus infections"; we are then talking about subacute and chronic disease, slowly but unremitting progressive disease, and recurrent disease in which the virus is persistent and the tissue damage is cumulative. Viruses which may be so persistent are decidedly not "slow" viruses in vitro or in the appropriate susceptible host. Thus, viruses such as herpes simplex virus and rubella virus have nothing slow about their behavior in a susceptible primarily-infected host. They produce classical examples of acute infections with rapidly replicating agents, which in some cell systems may be cytolytic. Even influenza, mumps, measles, rabies, and many other viruses causing acute diseases may be induced to form symbiotic, persistent infections in certain cell systems in vitro. 321 T o turn to a problem of semantics and terminology, we named our section eight years ago a "Laboratory of Slow, Latent, and Temperate Virus Infections"—a bit facetiously. At that time we knew of no viral disease in man in which a "temperate" relationship between the virus and the human cell had been established. On the other hand, it has been shown that in their oncogenic roles the SV40 virus and some of the adenoviruses can enter into a genetic, genome-localized, "temperate" relationship with the cell. The work of Dr. Wallace Rowe and others has beautifully documented this. Thus, the analogies which were drawn between mammalian viruses and bacteriophage in the early days of phage work are now supported fully by dynamic studies of mammalian virus-cell interaction. Many terms for this sort of infection are being bantered about. (Parenthetically, I may add that I am sure no three virologists would agree on a definition of these terms as applied to a mammalian virus or a mammalian cell system.) We talk of "persistent" or "endosymbiotic" infections; of slow, latent, and temperate infections; of masked, hidden, eclipse-phase; integrated, lysogenic, and repressed viruses; and also use their antonyms: vegetative as opposed to integrated; and derepressed versus repressed; partial, imperfect, incomplete, defective, and incompletely assembled viruses as opposed to complete or fully formed, or infectious virions. T h a t these mechanisms are involved in any given human infectious disease has yet to be fully established. The terms usually are taken from a much more quantitative and exact science of bacteriophage virology. Even now, in oncogenic virus work we refer to "permissive" and "nonpermissive" cells which are "competent" or "incompetent." T h e semantics eventually will be worked out. No one should imply that all other mammalian virologists agree with him in the way he 322 GAJDUSEK A.J.C.P.—Vol. 56 ENAGE 1 AUG 63 t XL" brain ic AUG 63 brain ic iv DEC 68 T SEPT 63 • Chimpanzee A1f Spider monkey S45f Chimpanzee A2m inc: 21 mot. dur: 9 mot. inc. 23 mot. dur: 1}4 mot. inc. 30 mo*. dur: 4 mos. Chimpanzee A4f inc: 20 mos. dur: 5 mos. IGIERAKABA — i — Chimpanzee A17m inc: 16 mot. dur: 5 mot. Chimpanzee AlBm inc: 17 mot. dur: -6 mos. Chimpanzee A19m inc: 19 mos. dur: 7 mos. KABUINAMPA brain brain iciv NOV 63 _1 Chimpanzee A5f Chimpanzee A6f Chimpanzee A7m Chimpanzee A8f inc: 28 mos. dur: 4 mos. inc: 27 mos. dur: 4 mos. inc: 25 mos. dur: 5 mos. inc: 38 mot. dur: 6 mos. inc: 19 mot. dur: 6 mot. Chimpanzee A43m inc: 39 mos. dur: 2 mos. inc. 18 mos. dur: 8 mos. TO"1 220 nm ic iv 85° C/30 mint, iciv T Chimpanzee A46m Chimpanzee A9f Capuchin monkey 666Wm t Chimpanzee A93m inc: 45 mos. dur: 1 mo. inc: 14 mos. dur: 2 mos. Chimpanzee A25m Chimpanzee A47m inc. 17 mos. dur: 3 mos. inc: 18 mot. dur: 6 mot. 10'° icip ivsc im r Chimpanzee A90m Chimpanzee A92m inc: 24 mos. dur: 1 mo. inc: 18 mot. dur: 1 mo. Squirrel monkey SSCIm inc: 25 mos. dur: IK mos Fie. 1. Transmission of kuru directly from human brain tissue of 11 different patients to chimpanzees and to New-World monkeys, with incubation periods varying from 14 to 45 months. T h e brain tissue of patient Kupenota (lower right) produced disease in chimpanzee A92 when inoculated at 10"B dilution. is using any of these terms, unless he defines his usage carefully. The disease I pick for brief description is kuru. Dr. Vincent Zigas and I first reported kuru in 1957 and have worked on this disease continually since that discovery. We may soon be reporting its demise and disappearance. None of you, and indeed very few physicians throughout the world, have had a chance to see patients with kuru. Yet, in its decade and a half of medical history, more than 300 scientific papers have dealt with it and whole symposia have been devoted to it in London, Paris, Washington, and Port Moresby. Kuru is an exotic, subacute, progressive, degenerative disease of the central nervous system in a highland neolithic population in New Guinea, a cannibalistic Stone Age group in which we discovered the disease, new to Western medicine, 13 years ago.12 It certainly was that disease of man which brought slow virus infections of man to worldwide consideration. It is the first chronic degenerative disease of man with a clearly established viral etiology, transmissibility, and excellent animal model. I can now say "models" since the disease is transmissible to several hosts.9 Furthermore, the cause of the disease in the animal models is certainly the same as the cause of the disease in man. I will return to kuru, but to advance the discussion quickly I will point out that its close pathologic and clinical similarities to scrapie, a slow virus disease of sheep, led to our transmission of kuru to chimpanzees, spider monkeys, squirrel monkeys, and the capuchin monkey, with incubation periods of \y2 to 4y2 years. This degenerative, September 1971 323 SLOW VIRUS DISEASES OF T H E CNS spongiform encephalopathy of man, which is a system disease of the cerebellum, presenting clinically with incoordination, truncal ataxia, tremors, dysarthria, and progressive motor incoordination, and which in all cases is fatal, usually within $ to 9 months, has now led us to discover quite similar disorders throughout the civilized world, perhaps caused by very similar agents—certainly, again, viruses. As a consequence of our studies of kuru, we have pursued further pathologic and clinical analogies and studied the presenile dementias which, in the Creutzfeldt-Jakob form, present a neuropathology quite similar to that we see in kuru in man, chimpanzees, and New World monkeys which develop the disease. We were looking for a less exotic and less geographically restricted disease in man which has a similar, strange pathology. This pathology cannot be easily produced by artifacts; a spongiosis of gray matter with intracellular vacuolation of neurons, a generalized gliosis, and a system degeneration pattern of neuron loss. We have now successfully transmitted CreutzfeldtJakob (C-j) disease from 11 humans to 13 chimpanzees inoculated with suspensions of brain tissues. Each of the chimpanzees developed a clinical disease and neuropathology similar to those seen in the human patients. We have inoculated animals witli brain and visceral tissues obtained by surgical biopsy or autopsy from many additional cases of this disease that have occurred throughout the United States, Canada, Europe, and Australia. Most specimens have been inoculated into New World monkeys (Fig. 1) and chimpanzees (Fig. 2), A.T. M.W. -I biopsy brain 5% ic iv autopsy brain I __l NOV 66 10% ic iv FEB 68 20% ip iv im MAY 69 autopsy brain 10% ic iv FEB 68 autopsy brain 10/S ic iv MAR 68 Chimpanzee A54 m Chimpanzee A79f Chimpanzee A77f Chimpanzee A124m Chimpanzee A78f Chimpanzee A81 m inc: 13 mos. dur: 1mo. inc 12 mos. dur 2J4 mos. inc: 12 mos. dur: Yh mos. inc: 16 mos. dur: 2 mos. inc 14 mos. dur 2 mos. inc: 14 mos. dur: 1 mo. J.J. E.S. autopsy brain 10% biopsy brain 5% autopsy + biopsy brain 10% J.D. L.E. D. M. autopsy brain 10% .autopsy brain 5%" autopsy brain 5% IC IV IC IV ICIV IC IV IC IV IC IV JUNE 68 AUG 68 FEB 69 MAY 69 OCT 69 MAR 70 Chimpanzee A106m Chimpanzee A114f Chimpanzee A119m Chimpanzee A123m Chimpanzee A142 f Chimpanzee A152m inc: 13 mos. dur: 1 mo. inc: 12 mos. dur: 1 mo. inc: 12 mos. dur: V/i mos. inc: 11 mos. dur: 2 mos. inc: 14 mos. dur: 1 mo. inc: 11 mos dur: 1 mo. FIG. 2. Transmission of Creutzfeldt-Jakob disease from 10 human patients to chimpanzees, with incubation periods of 11 to 16 months. T h e brain of patient M. W. (top row center) produced disease in chimpanzee A124 when inoculated intraperitoncally, intravenously and intramuscularly, without intracerebral inoculation. 324 GAJDUSEK but sufficiently long incubation periods have not yet passed to report any of these experiments as negative. Incubation periods observed in chimpanzees that have developed C-J disease have ranged from 11 to 14 months. On second passage in chimpanzees the incubation period has not changed. The disease in all susceptible species of subhuman primates is similar to Creutzfeldt-Jakob disease in man. All clinical features are present: myoclonus, fasciculations, spasticity, and apparent dementia. Most strikingly, the neuropathologic lesions are identical.4- 20>21 Indeed, our neuropathologist collaborators now claim that they cannot distinguish at the cellular level C-J disease pathology of a chimpanzee from a human brain with either kuru or Creutzfeldt-Jakob disease. We no longer consider kuru an exotic, isolated, strange disease which a few physicians have reported only from New Guinea, but rather, a type-disease which is found to be mimicked in every major city in the world, in the form of Creutzfeldt-Jakob presenile dementias. Although most pathologists clearly distinguish the Creutzfeldt-Jakob type of presenile dementia from the more common Alzheimer type, some cases are borderline between the two in their clinical or pathologic pictures and, at times, in both. With two viruses of man (kuru and Creutzfeldt-Jakob disease) causing slow formation of amyloid-containing plaques, similar to Alzheimer's and senile plaques, and a slow generalized neuronal loss with gliosis, we have some of the features of the aging brain produced by a slow virus infection. 8 Whether immune mechanisms are involved in the pathogenesis of these diseases, we cannot yet specify, since no serologic typing of these viruses or, for that matter the related diseases, scrapie and mink encephalopathy, has yet been possible. Yet as kuru was being elucidated, my own laboratory and several others were simultaneously working on other diffuse A.J.C.P.—Vol. 56 subacute degenerations of the brain that strongly suggested to us the possibility of a slow virus infection. One of these suddenly provided clues showing that it was, in fact, caused by such an infection: namely, Dawson's or subacute type A inclusion body encephalitis, called, in Europe, Van Bogaert's subacute sclerosing leukoencephalitis, or Pette-Dbring panencephalitis. Doctors Zeman, Sever, Alpers, Adels, Gibbs, and I decided to rename the disease because of its many different eponyms and other synonyms when we were preparing for the world conference we convened on the disease three years ago at the National Institutes of Health. 25 At that time, measles virus had become heavily implicated as the etiologic agent, in several laboratories including my own. We had to give the disease a new and descriptive name to surmount the huge synonymy, but we were unable to make a good choice. We settled in compromise on "subacute sclerosing panencephalitis," or "SSPE," which the disease is now generally called. It is a bad name. We had better have called the disease "delayed and slow measles encephalitis." In this disease of children and young adults between the ages of 3 years and their early 20's (most common in children less than 10 years old), there is progressive dementia, myoclonic jerking, and an afebrile course of progressive motor disability, and increasing neurologic deterioration almost always fatal in 1 to 3 years. Typical periodic slow-wave discharges are found in the EEG, and the cerebrospinal fluid shows a paretic colloidal gold curve and elevated gamma globulin. This disease gives us a convincing case for persistent virus-induced autoimmune damage in man. Measles virus has now been isolated repeatedly from brain biopsies and autopsy brain tissue of these patients. It follows the primary episode of acute measles, often in early infancy, by a period ranging from a few months to more than 10 years. Many patients have histories of September 1971 SLOW VIRUS DISEASES OF T H E CNS atypical measles, measles without rash, or measles exposure but no diagnosed measles. It is not the same as classical post-measles encephalitis; rather, it is a delayed, slow measles encephalitis with a progressive course ranging from a few months to several years to death. In SSPE, the final isolation of the infectious measles virus was slow in being achieved, although we had ample indirect evidence that it was the cause. Its study provides a lesson for the investigation of any disease which may have a persistent, latent viral etiology. We had extended to SSPE our origfnal approach to kuru and begun to inoculate chimpanzees and other hosts using the "Noah" approach: two or more of every species of subhuman primate we could obtain were inoculated and held under observation. We had done this with brain biopsy and early autopsy material from several cases of SSPE obtained for us from European and American patients by cooperating neurosurgeons and neurologists. However, no disease developed in any of the animals inoculated over long-term observation. Yet, SSPE could not be removed from the 20 idiopathic, degenerative diseases of the central nervous system of man which we compiled in 1958 as demanding this sort of search for a transmissible agent. Then the work of French electron microscopists, Bouteille and co-workers,5 was brought to our attention, work in which they reported finding tubular structures in the intranuclear inclusion bodies in this disease, which suggested to them myxoviruses. Others claimed that they were merely neurotubular structures of a type not unusual in degenerating neurons. We immediately recognized that it is probably expecting too much for a common myxovirus infection of man to be neurovirulent in our exotic hosts. Thus, we started virus serologic study of our SSPE patients and inoculation of animals and tissue and cell cultures in attempts to isolate measles virus 325 from the tissues we had received from patients with the disease.1 Connolly and his co-workers0 in Belfast detected unusually high measles antibody titers in the serum and cerebrospinal fluid (CSF) of two patients and by direct FA test demonstrated fluorescence of material in intranuclear inclusion bodies in the patient's brain when stained with convalescent measles serum that had been conjugated with isothiocyanate. We quickly confirmed these findings in our laboratory, and at the same time we demonstrated the specificity of the antibody in the patient's serum and CSF to measles virus with no unusual serologic reactions or cross-reactions with other myxovirus antigens. 1 That SSPE is, in part at least, an autoimmune disease, is suggested by the following. There is uniformly a hyperimmune serum response to measles virus antigens. Specific antimeasles gamma globulin is found in the spinal fluid. There is a plasma cell proliferation in the brain with mononuclear cell proliferation producing a perivascular cuffing. Antibody is produced in the brain itself by plasma cells in the inflammatory reaction. There is fluorescent staining of the SSPE brain sections using anti-gamma globulin and also anti-beta C'3 (third component of complement) according to the technic of Dixon and Oldstone. 7 This indicates that an antigen has coupled to the specific antibody in these cytoplasmic areas; ter Meulen, 28 using quickly frozen brain obtained immediately after the death of a child who had been carefully studied during the course of typical SSPE, succeeded in demonstrating that the gamma globulin extracted from washed brain fragments was measles specific antibody and not antibody to some other myxovirus. The gamma globulin content of the brain was quantitatively so high that it resulted in a very significantly higher yield of globulins from brain tissue than could be obtained from normal brain. Once the measles anti- 326 GAJDUSEK body had been eluted from frozen lyophilized brain sections with 3 M thiocyanate buffer of pH 6.0, measles antigen in the cytoplasm was unmasked. Fluorescent staining of antigen in the cytoplasm with measles specific antiserum was then possible. Without such pretreatment to release and unmask the measles antigen from gamma globulin antibody binding, only intranuclear inclusions were stained with fluorescein coupled measles antisera. Negative controls were obtained using other virus specific antibodies in direct and indirect FA systems. Do we have an experimental animal model for the study of this disease? No. In addition to the work we have been doing, many other attempts have been made to transmit the disease to ferrets, monkeys, and other laboratory animals, using as inocula brain and other tissue suspensions from patients with SSPE as well as the tissue culture isolated measles virus. Katz and his co-workers17 have reported an inapparent or subclinical infection of ferrets manifested by EEG changes and a mild encephalopathy following inoculation of SSPE brain suspensions. However, no disease lending itself to therapeutic trials is produced. Long before SSPE was known to be associated with measles virus, Rustigian 23>2i succeeded, in a laboratory exercise which has been repeated, in modifying the measles virus through passage in tissue culture, such that it no longer is cytolytic, but becomes completely parasymbiotic. The measles virus now, in a repressed, incompletely assembled form, filled the cells maintained in vitro, passing from cell to cell, and yet was not being released to the outside medium as a hemagglutinin or an infectious virus particle. T h e procedures found necessary in isolating the measles virus regularly from the biopsies of brains of SSPE patients is revealing. The fresh brain tissue is minced A.J.C.P.—Vol. 56 into small fragments, trypsinized, and the trypsin-dispersed cells are layered in flasks to produce cell cultures. One may also grow the brain tissue as an explant, using pinhead-sized fragments. In either case, some brain cells, principally astroglia, grow well. They may continue to survive and slowly divide for weeks or many months. But no measles virus is usually released, there is no cell surface hemagglutinin, and there is no FA-positive reaction on either the trypsinized or explant cultures, at first. However, after prolonged cultivation in vitro, occasional giant cells appear; these are found to contain measles antigen by FA staining, and myxovirus microtubules by electron microscopy. 3 -" Furthermore, when these cells are subcultured or cocultivated with other cell lines, such as primary rhesus monkey kidney, stable green monkey kidney B-SC-1, and HeLa cells, they often yield polykaryons, and in an occasional polykaryon, with immunofluorescent technics, one can detect measles antigen at a time when no hemagglutinating fully infectious measles virus is detectable. There is no infectious virus released into the medium and there is no infection caused by these cultures, at this stage, to a measles susceptible system. However, by repeated passages of persistently infected cells and continued subculturing of polykaryons, that is, human brain cells fused with measles virus permissive cells, fully infectious active measles virus is shed, which on genetic analysis turns out to be intermediate in its properties between vaccine strains and the wild virus of natural acute measles.14 All the trypsinization and other manipulations through many subcultures that have gone into the process of viral genome rescue, or unmasking of the virus, would certainly be sufficient to accomplish a genetic alteration of the strain isolated. Therefore, the answer to the question, "What is the relationship of this isolated measles virus to wild measles or attenuated live measles September 1971 SLOW VIRUS DISEASES OF THE CNS vaccine strains?" is not readily answerable, since the modification that isolation technics caused cannot be predicted. As recent work by ter Meulen indicates, we undoubtedly have in SSPE, a situation in which the virus is not present as fully infectious or fully formed measles virions. Instead, there is an asynchronous, incomplete, and uncoordinated production of viral subunits; tubular structures without nucleic acid or nucleic acid and viral antigen, without full assembly of EM visible virions. This goes on in the human brain, spreading from cell to cell, as it occurs in model cultures infected with Rustigian's strain of defective, incomplete measles virus. That this kind of infection may go on for weeks, months, and years, perhaps decades, silently, without producing significant pathology and then, for unknown reasons, may be triggered into a more rapid, slowly pathogenic course, is the significant fact for consideration in the study of chronic idiopathic disease. Whether a virus specific defect in the delayed hypersensitivity reactions may contribute to such release of latent virus is not yet established. However, the genetic heterogeneity of the population with respect to immunologic competence may well account for a rare individual whose delayed or sluggish immune responses favor the development of such a measles infection. Finally, we have a model of this disease in nature, in hard-pad disease in the dog. The closest virus serologically to measles is the distemper virus. It is not distemper virus in the human brain causing SSPE, but measles. However, dogs with distemper occasionally recover, and a small percentage develop a neurologic disease with myoclonic jerking and progressive cerebral degeneration, quite akin to SSPE. This spontaneous, natural disease is not a good laboratory model for studying SSPE, since we cannot produce the disease in more than a rare animal that recovers from distemper. 327 Only after intranasal infection with the distemper virus has the incidence of this complication been raised, but still not sufficiently to make it a usable system for experimental trial therapy and other studies. In these studies half of the dogs died of their primary distemper infection and only half of the survivors went on to develop a chronic CNS disease. Interestingly, this syndrome occurred only in those dogs which developed delayed antibody responses in their primary distemper infection. If antibody formation occurred early enough to prevent spread of virus from lymphatic cells to epithelial cells, these tissues were not infected. If virus reached such cells as epidermal cells and neurons before protective levels of antibody were attained, the virus persisted and late encephalitis, or hard-pad disease developed.2 Koprowski and associates18 have found in explant cultures of SSPE brain tissue, grown for weeks in in vitro cultures, a papova virus identified by electron microscopy study. It is neither polyoma nor SV40, but in size and morphology is indistinguishable from these known viruses. Neither is it the rat papova viruses, nor any of the morphologically similar viruses such as the virus of warts. They hypothesize that this virus is so often found by electron microscopy in the SSPE explant cultures that it, in addition to the proved presence of measles virus, must be involved in the pathogenesis of the disease and, further, the two viruses must act symbiotically, and the papova virus must stimulate the disease produced by the measles virus. We do not know what to call this papova virus. I suspect that it is the same virus that ZuRhein and Chou 2 7 and others have been finding regularly in the lesions of progressive multifocal leukoencephalopathy, which they have similarly been unable to distinguish morphologically from polyoma or SV40 viruses, and which they and we have been unable 328 GAJDUSEK to cultivate in vitro* I tend to call it "the papova virus latent in human brain." Progressive multifocal leukoencephalopathy (PML) is a fatal brain disease which develops in patients who are dying of chronic Hodgkin's disease, Boeck's sarcoid, tuberculosis, leukemia, or other lymphomas, and in whom the subacute brain infection kills them before their primary disease. At autopsy one finds pinpoint scattered lesions in the white matter of the brain which are not, on pathologic examination, caused by the primary disease. Since the lesions are filled in their peripheries with crystalline arrays of papova virions, we have a good idea of what has happened: a latent agent has been activated in the course of the immunosuppression that accompanies these natural diseases. Since ZuRhein's first demonstration of intranuclear crystalline masses of papova virus in the pinpoint lesions of this disease, others have repeatedly confirmed her finding, but the virus still has not been identified. We have, with the aid of Dr. Wallace Rowe of the NIH, found no evidence of antibodies to SV40, polyoma, or to the papova rat (RV and X14) viruses, or mouse virus (K virus) in sera from the patients, or from animals inoculated with this disease. How many other viruses can man silently carry for years, like herpes simplex, measles virus, infectious hepatitis, rubella virus, cytomegalovirus, the EB virus of infectious mononucleosis, the varicella virus, warts, adenoviruses, or reoviruses? How many other agents of this sort do we have lying in wait in our tissues for the right additional factors to appear to stimulate them to path* More recently, this virus has been cultivated in three laboratories. [Gardner SD, Field AM, Coleman DV, Hulme B; New human papovavirus (B.K.) isolated from urine after renal transplantation. Lancet 1:7712, 1253-1256, 1971. Padgett BL, Walker DL, ZuRhein GM, Eckroade RJ; Cultivation of papovalike virus from human brain with progessive multifocal leukoencephalopathy. Lancet 1:7712, 12571260, 1971] A.J.C.P.—Vol. 56 ogenic activity? Such factors may be the presence of a second virus from a new acute infection or another persistent agent; they could be a bacterial or a protozoal or helminthic infection, or the immunosuppressive effects of another chronic disease or that induced therapeutically for organ transplantation, or they might even be provided by certain drugs, toxins, nutritional or dietary factors or, finally, even traumatic injury or changes in endocrine balance. Indeed, these may be the underlying mechanisms of many chronic diseases of unknown etiology. That such latent and persistent infections may eventually produce a hyperimmune state and lead to autoimmune tissue damage with the antibody reacting with the virus filled tissue cells is amply demonstrated in SSPE in man and in the persistent-tolerant infection of mice with LCM virus which develop immunecomplex renal disease. Finally, such a mechanism is not even excluded in diseases known to be heredofamilial, for the genetic abnormality of the immune system may well predispose to autoimmune disease in the presence of persistent virus infections, as appears to be the case with the NZB mice. Now to return to kuru. The disease has been transmitted to more than 50 chimpanzees (another 30 have been given Creutzfeldt-Jakob disease).13 Kuru is in the fifth passage in the chimpanzee, and in the third passage in the spider monkey. In the fourth passage, the virus reaches titers of 107 infectious doses per ml. in the brain, which is as high as some of the equine encephalitis virus titers attained in human brain. It is present also in the lymph nodes and visceral tissue, as well as the brains of affected chimpanzees, and can be transmitted to the chimpanzee by peripheral, non-intracerebral, routes of inoculation. No electron microscopically visible particles identifiable as virions have been found in any of our studies of either kuru September 1971 329 SLOW VIRUS DISEASES OF T H E CNS or Creutzfeldt-Jakob disease; instead, only stacks of curled fragments of plasma membrane filling the intracellular vacuoles of neurons have been observed.20-21 Our search for virus which we can cultivate in vitro from the surgically sterile tissues of animals affected with kuru and Creutzfeldt-Jakob disease has yielded more than 150 isolations of virus strains from tissues which are bacteriologically and virologically sterile for the first weeks of study. They are thus truly "latent" viruses. Attempts to isolate virus by inoculating grindings of the fresh tissue into many primary and continuous cell lines have been negative. However, by maintaining explant cultures of brain, thymus, lymph nodes, kidney, etc., for weeks, months, and as long as a year, my colleagues have been isolating strains of latent viruses from these "sterile" explains. 11 ' 22 These many isolated chimpanzee virus strains have been typed into 13 different viruses. There are few fragments of chimpanzee brain, from young or old animals, from which we have not isolated viruses. Sometimes, not only one virus, but viruses, i.e., two or more strains from one explant, have been isolated. We cannot say this yet about human tissues as well. We have isolated only a few agents from human tissue culture explants thus far. This discovery of latent viruses in "sterile" tissues should not have surprised us, had we just thought about the history of poliovirus vaccine. We used millions of doses of a virus vaccine which we never would have started to use, had we thought it contained either live or dead extraneous unidentified virions. Yet, within a few years of its introduction, there were dozens of viruses known to be contaminating various lots of that vaccine. The simian viruses, one after another, were isolated from the healthy, surgically removed, at first virologically sterile, monkey kidney. This has been well known for years. One of the la- tent simian viruses, SV40, was even distributed live from the drug store shelves, uninactivated by the concentration of formalin used to inactivate the poliovirus. However, we continued to think "the kidney is a 'dirty' organ; the monkey a 'dirty' animal." Our more recent work with the chimpanzees affected with kuru indicates that their brain and thyroid are equally "dirty." Such latency can, of course, suggest possibilities for the triggering of autoimmune disease. Such virus infections may lead to the alteration of cell-surface antigens and the addition of new cell-surface antigens and of new intracellular antigens. These new antigens may be, at times, virus subunits which are not being assembled into complete virions—defective viruses, if you wish—although the whole genome is present, some of it repressed, and assembly is not proceeding normally. The latent virus genome may also program for the production of non-virus antigens, such as tumor antigens, and thus produce antigenic molecules which are not virus subunits. We are thus immediately faced with the possibility of autoimmune mechanisms at work in cancer as well. I leave the matter of such speculations here, and with apologies, I will not go into the long and involved story of kuru and its possible origins at this time. References 1. Adds BR, Gajclusck DC, Gibbs CJ Jr, et al.\ Attempts to transmit subacute sclerosing panencephalitis and isolate a measles related agent, with a study of the immune response in patients and experimental animals. Neurology 18:1 (part 2), 30-51, 1968 2. Appcl MJG: Pathogenesis of canine distemper. Amer J Vet Res 30:7, 1167-1182, 1969 3. Baublis JV, Payne FE: Measles antigen and syncytium formation in brain cell cultures from subacute sclerosing panencephalitis (SSPE). Proc Soc Exp Biol Med 129:593-597, 1968 4. Beck E, Daniel PM, Gajdusek DC, et at.: Subacute degenerations of the brain transmissible to experimental animals: A neuropathological evaluation. Proc. Vlth Congr. Inter, de Neuropathologie. Paris, Masson et Cie, 1970, pp 858-873 330 GAJDUSEK 5. Boutcille M, Fontaine C, Vedrenne C, et al.: Sur un cas d'enccphalite subaigue a inclusions. Etude anatomo-clinique et ultrastructurale. Rev Neurol 113:4, 454-458, 1965 6. Connolly GH, Allen IV, Hurwitz LJ, et al.: Measles virus antibody and antigen in subacute sclerosing panencephalitis. Lancet 1:542, 1967 7. Dixon FR, Oldstone MBA: Lymphocytic choriomeningitis: Production of antibody by "tolerant" infected mice. Science 158:1193-1195, 1967 8. Gajdusek DC: Slow virus infections and activation of latent virus infections in aging. Advances Geront Res (in press), 1971 9. Gajdusek DC, Gibbs CJ Jr: Transmission of two subacute spongiform encephalopathies of man (kuru and Creutzfeldt-Jakob disease) to New World monkeys. Nature 230:588-591, 1971 10. Gajdusek DC, Gibbs CJ Jr, Alpers M (editors): Slow, Latent and Temperate Virus Infections. NINDB Monograph No. 2, PHS Publication No. 1378, U. S. Government Printing Office, Washington, D. C , 1965, 489 pp 11. Gajdusek DC, Rogers NG, Basnight M, et al.: Transmission experiments with kuru in chimpanzees and the isolation of latent viruses from the explanted tissues of affected animals. Ann NY Acad Sci 162:1, 529-550, 1969; also, Virology Abstracts, V3484, 1969 12. Gajdusek DC, Zigas V: Degenerative disease of the central nervous system in New Guinea. T h e endemic occurrence of "kuru" in the native population. New Eng J Med 257:30, 974978, 1957 13. Gibbs CJ Jr, Gajdusek DC: Transmission and characterization of the agents of spongiform virus encephalopathies: Kuru, CreutzfeldtJakob disease, scrapie and mink encephalopathy, Immunological Disorders of the Nervous System. Baltimore, Williams and Wilkins, A.R.N.M.D. 49 (in press): 1971 14. Horta-Barbosa L, Fuccillo DA, Hamilton R, et al.: Some characteristics of SSPE measles virus. Proc Soc Exp Biol Med 134:1, 17-21, 1970 15. Horta-Barbosa L, Fuccillo DA, London W T , et al.: Isolation of measles virus from brain cell cultures of two patients with subacute sclerosing panencephalitis. Proc Soc Exp Biol Med 132:272-277, 1969 16. Johnson R T , Merger EH: Experimental rabies: Studies of cellular vulnerability and pathogenesis using fluorescent antibody staining. J Neuropath Exp Neurol 24:662-674, 1965 A.J.C.P.—Vol. 56 17. Katz M, Rorke LB, Masland WS, et al.: Transmission of an encephalitogenic agent from brains of patients with subacute sclerosing panencephalitis to ferrets. New Eng J Med 279:793-798, 1968 18. Kibrick S, Gooding GW: Pathogenesis of infection with herpes simplex virus with special reference to nervous tissue, NINDB Monograph No. 2, Slow, Latent, and Temperate Virus Infections. Edited by DC Gajdusek, CJ Gibbs Jr, and M Alpers. U. S. Government Printing Office, Washington, D. C , 1965, pp 143-154 19. Koprowski H, Barbanti-Brodano G, Katz M: Interactions between papova-like virus and paramyxovirus in human brain cells: A hypothesis. Nature 225:1045-1047, 1970 20. Lampert PW, Earle KM, Gibbs CJ Jr, et al.: Electron microscopic studies on experimental spongiform encephalopathies (kuru and Creutzfeldt-Jakob disease) in chimpanzees. Proc. Vlth Congr. Intern, de Neuropathologie, Paris, Masson et Cie, 1970, pp 916-930 21. Lampert PW, Gajdusek DC, Gibbs CJ, Jr: Experimental spongiform encephalopathy (Creutzfeldt-Jakob disease) in chimpanzees: Electron microscopic studies. J Neuropath Exp Neurol 30:1, 20-32, 1971 22. Rogers NG, Basnight M, Gibbs CJ Jr, et al.: Latent viruses in chimpanzees with experimental kuru. Nature 216:446-449, 1967 23. Rustigian R: Persistent infection of cells in culture by measles virus. I. Development and characteristics of HeLa sublines persistently infected with complete virus. J Bact 92:1792, 1966a 24. Rustigian R: Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa sublines and recovery of a HeLa clonal line persistently infected with incomplete virus. J Bact 92:1805, 1966b 25. Sever JL, Zeman W (editors) : Conference on "Measles Virus and Subacute Sclerosing Panencephalitis." Neurology 18:1 (Part 2), 1-200, 1968 26. ter Meulen V, Enders-Rucklc G, Miiller D, et al.: Immunohistological, microscopical and neurochemical studies on encephalitides. III. Subacute progressive panencephalitis, virological and immunohistological studies. Acta Neuropath 12:244-259, 1969 27. ZuRhein GM, Chou SM: Papova virus in progressive multifocal leukoencephalopathy, Infections of the Nervous System. Baltimore, Williams and Wilkins, A.R.N.M.D. 64:307362, 1968 Discussion following Dr. Gajdusek's Paper (In response to question from the audience) DR. GAJDUSEK: At the Wistar Institute, Doctors Hilary Koprowski and Michael Katz have been able to produce an EEG pattern in ferrets inoculated with suspensions of brain from patients with SSPE similar to that seen in the patients. When the apparently healthy animal is sacrificed they show an inclusion-body type of panencephalitis resembling that of SSPE. When they take the isolated measles virus together September 1971 SLOW VIRUS DISEASES OF THE CNS with cells in which polyoma-like virus particles are visible by electron microscopy (which is the only way it can be demonstrated), this mixture of the two agents produces the phenomenon more regularly in ferrets, and now in hamsters as well. I have purposely avoided the complex problem of how one is ever, in chronic persistent incomplete virus infections, going to fully investigate the matter of possible viral pathogenesis. It is better here, to an audience rather than in press, to become wildly speculative. If we accept the fact that defective, unassembled replication of viruses continues over years silently, and then, spontaneously, from factors we do not understand, can be stimulated into a different level of misbehavior so that disease with a cumulative, finally fatal pathology results; if we accept this as a fact for measles virus in SSPE and distemper virus in hard-pad disease—what is then the next analogy we can make with defective bacteriophage systems? We can have more extensive defectiveness; namely, the genetic information for the whole virion is not really present, but masked or repressed. Assembly may not be proceeding at all. The equivalent of only a tumor antigen is left and no infectious virions are resurrectable under any conditions. If that sort of thing occurs, and it surely does go on in experimental models in a test tube, it might be such a mechanism that lies behind Parkinson's disease with a defective replication of influenza virus, multiple sclerosis with a fully defective and incomplete bit of measles virus, or hepatitis or some other virus. Such theories are effortless armchair exercises; what we need more is a means of probing in these directions. How are we going to get at it? The only approaches currently available, I'm afraid, are to make a potent antisera against each of the subviral components of the whole gamut of myxoviruses and other possible virus candidates for use against appropriately collected tissue specimens. Then 331 you, as clinical pathologists, collect material, not in formalin but in dimethylbutane and gluteraldehyde, and quick-frozen thin sections at —70 C. and liquid nitrogen temperatures. With the bulk of such tissue we may also be able to make density gradient fractions of viral subunits in the tissues. This should be the approach in any idiopathic disease these days. We may forget the old hematoxylin-and-eosin stained section. For chronic degenerative central nervous system diseases the neuropathologists, neurosurgeons, and neurologists are supplying us with well collected tissues in large amounts and very often. We are working intensively on rare diseases: Pick's, Alzheimer's, metachromatic leukodystrophy, Huntington's chorea, myoclonic epilepsy, and other rare neurologic syndromes. Tissues from many cases of each of these diseases are being accumulated as brain biopsies and as specimens from autopsies within a few hours of death. We are having less luck with the more common diseases, such as multiple sclerosis and disseminated lupus erythematosus. We need live cells for trypsinized cultivation. We do not need material taken five hours postmortem and put into a fixative, but material collected immediately, within a few hours of death, in a sterile manner. When it is taken from separate organs, the autopsy room must be equipped with separate sets of sterile instruments and containers. There is no point in opening the head with one set of instruments, and then taking specimens of liver and spleen with the same instruments. Most autopsy rooms are not even equipped with separate sets of sterile instruments and other equipment for collecting sterile specimens. So I make a real plea. If, within the field of rheumatoid diseases, the collagen diseases, the other degenerative idiopathic diseases in addition to the neurologic, you are planning to follow the immunologic and virologic investigative approach, reequip the autopsy and the surgical biopsy 332 GAJDUSEK rooms for the sterile collection of proper specimens, some quickly frozen in liquid nitrogen, others in other special tissue culture media for cultivation in tissue cultures. (In response to question from the audience) DR. GAJDUSEK: The whole discipline of virology depends on serologic identification and the specificity of the serologic reactions. In other words, when you make the diagnosis in any virus diagnostic laboratory of poliomyelitis or any other specific virus infection, you rarely do anything but decide what virus antigens or antibodies are there, depending on the specificity of serologic reactions. If we discard this, we have discarded neutralization, complement fixation, hemagglutination-inhibition, fluorescent antibody, and every other serologic diagnostic method. In other words, when I identify rabies or poliomyelitis viruses in the laboratory, I can assure you that it is rabies or poliomyelitis, respectively, by specific antigen-antibody reactions, or using these serologic procedures state that it is an unknown agent which is related antigenically to the known viruses. Now, in the case of the measles virus in SSPE, we are growing the infectious measles virus from the brain. So the full genetic information of the measles virus is there. It takes great effort to isolate it, however. We first thought that this was because we had to overcome the neutralization of the virus which already had occurred in vivo. On the other hand, dilution does not release the virus, and it is obtained only when we produce cell fusions. When that trick works, as it does in many other experimental viral systems, we are inclined to think the virus A.J.C.P.—Vol. 56 was in a genetic modification well synchronized with the host and no longer producing cytolytic effect. The antigen present has not been identified. It may be coat antigen, or something else. The antisera which have been made and which have worked are crude antisera made against a tissue culture harvest of measles virus— not even the purified measles virus. Until we have a more accurate antigenic tool— antisera against component parts of measles virus—I do not think we can answer the question of participation of the various parts of the virus in the disease process. (In response to questions from the audience) DR. GAJDUSEK: One may ask of a surgical biopsy of any organ: "Are there virus agents there? What state are they in? How many agents? Do they have any role in any pathology seen?" I think those are all fair questions. That the symbiotic relationship may be deranged and the latent viruses begin producing disease is already demonstrated in certain diseases. For example, Dr. Sydney Kibrick and his colleagues in Boston have regularly produced excretion of herpes simplex from corneal cells, followed in some animals by herpes encephalitis, by simply injecting adrenalin into normal animals which had stopped shedding herpes virus from their corneas for many months after they had been given corneal herpes simplex infections and recovered. His triggering shot to produce the shedding of infectious herpes simplex virus, new corneal pathology, and even acute or chronic herpes invasion of the nervous system, is an injection of adrenalin. 18