Transmissible spongiform encephalopathy:
In addition to the accurate clinical description of scrapie, two points merit special emphasis: first, scrapie was recognized as a contagious disease in sheep, and second, scrapie was not considered to be a human pathogen (at least, not for the lower classes). Nothing we have learned in the last 250 years has invalidated these observations.
Around the middle of the 19th century, veterinarians in England, France, and Germany initiated the scientific study of scrapie, including systematic neuropathologic examinations, and efforts to identify an infectious pathogen.
In particular, Besnoit and his colleagues in the Toulouse School of Veterinary Medicine recognized neuronal vacuolation as a characteristic feature , and also attempted to transmit the disease to healthy sheep by inoculation of brain and transfusion of blood from affected animals, and by cohabitation of symptomatic sheep with healthy sheep . The negative results they reported after observation periods of up to several months were certainly due to a failure to appreciate the extraordinarily long pre-symptomatic phase of infection, a failure that 70 years later would also thwart the first experimental attempts to transmit a human spongiform encephalopathy.
Undeterred by these results, the French veterinarian community continued to explore the infectious nature of scrapie, and at length Cuillé and Chelle, taking note of several epidemiological studies that pointed to incubation periods of 18 months or longer in naturally occurring disease, succeeded in 1936 in transmitting scrapie to two healthy sheep by intraocular inoculation of brain or spinal cord tissue from an affected animal . The incubation period between experimental inoculation and onset of disease varied from one to two years, shortest when inoculated into the brain, longest when using a peripheral route. In subsequent experiments, they also transmitted disease using intracerebral, epidural, and subcutaneous routes of infection, and using brain tissue passed through a bacterial exclusion filter.
In a grand historical irony, this landmark series of experiments was being confirmed at the same time in England as a result of an outbreak of scrapie in several hundred sheep that had been immunized against louping ill with a vaccine prepared from brain, spinal cord, and spleen tissue from sheep that were belatedly discovered to have been exposed to natural scrapie infection . The transmissible nature of the scrapie agent was thus established beyond any doubt, although debate about the interplay between contagion and host genetic factors continues to the present day.
Throughout the 1940’s and 1950’s, the accelerating pace of veterinary research yielded many new discoveries about the behavior of the causative agent: its distribution through the body after experimental and natural infection; its physical association with cell membranes; its susceptibility to host genetic factors; and its extraordinary resistance to standard methods of inactivation. Especially notable were two seemingly contradictory observations: Dickinson et al , using the methods of classical genetics, identified a gene in both natural and experimental infections that determined phenotypically different strains of scrapie; whereas Alper et al.  showed that infectivity survived a dose of ionizing radiation that was incompatible with the biologic integrity of nucleic acid, an observation that led to several theories about the agent being a membrane-bound ligand, a lipid-protein-polysaccharide complex, and even an unadorned protein. Finally, and by no means least important, the successful adaptation of the agent by Chandler to laboratory mice , elicited a collective sigh of relief from experimentalists who had until then been obliged to work exclusively with sheep and goats.
Then, in 1959, this endemic disease of sheep, unknown or ignored by medical science, was proposed by the American veterinarian Hadlow  to be analogous to a newly described disease of humans, called kuru, an epidemic neurological disorder found in the Eastern Highlands of Papua New Guinea, that two years earlier had been introduced to Western medicine by Gajdusek and Zigas . The reverberations from these remarkable insights are still being felt.
Pediatrician by training, virologist by experience, and genius by nature, Gajdusek had just finished working in Sir MacFarland Burnet’s laboratory in Australia when an opportunity arose to re-visit New Guinea. Once there, he and Zigas, a Baltic States expatriate working as a Medical Patrol Officer, went into the Highlands to have a first-hand look at kuru. Just what it was about this disease, restricted to a remote and isolated group of 15,000 people, that aroused Gajdusek’s instincts about its potential larger importance remains obscure, but during the next decade he conducted a wide-ranging campaign of scientific investigation, often in a running battle with the Australian colonial bureaucracy, which reacted rather peevishly to a perceived threat of "outside" medical exploitation of its own territory.
Genetic, endocrine, nutritional, and toxic causes were explored, and particular attention was given the possibility of an infectious origin associated with the practice of ritual endocannibalism, which, although without either laboratory or pathological support, was the theory favored by everyone from missionaries to bush pilots. All conceivable means of detecting an infectious agent were attempted, including the inoculation of monkeys, with a uniform absence of success after observation periods of two to three months. While these efforts were continuing, Hadlow’s observation was published, leading to an extension of experimental inoculations to chimpanzees kept under long-term surveillance, and in 1965, three chimpanzees developed kuru 18-21 months after having been inoculated intracerebrally with brain tissue from different kuru patients .
Well before this result was known, the neuropathologist Klatzo had conducted an exhaustive study of 12 kuru brains , commenting that they resembled only one other human disease with which he was familiar – Creutzfeldt-Jakob disease (CJD), first described in the early 1920’s by two German neurologists whose names comprise its eponym, and subsequently reported from time to time in a literature that suggested both its rarity and uncertain diagnostic features. CJD was duly inoculated into chimpanzees, and transmitted disease within 12-14 months . Like scrapie, kuru and CJD were subsequently adapted to laboratory rodents.
The years following these discoveries were consumed by studies of the physical and chemical properties of the infectious agent, its distribution and titer in tissues of infected animals, and its host range. While knowledge of the biology of the agent was thus advancing, knowledge of its molecular biology remained in a primitive state because of the technical difficulty of separating the infectious agent away from contaminating host components. Alper’s work had stongly suggested that nucleic acid was not needed for replication, but theories of protein-directed replication were merely untestable intellectual gymnastics without an alternative molecule to work with. This point was not lost on the American neurologist Prusiner, who correctly saw that further advances in the field were going to depend upon a vastly superior process of agent purification than was presently available. After several years of work, he finally succeeded, with the combined use of detergent extraction and limited proteinase digestion, in obtaining a highly purified infectious preparation that yielded an N-terminal peptide sequence sufficient for the corresponding cDNA to "fish out" the encoding nucleic acid of a full-length protein (PrP) . To the surprise of everyone, this coupling of biochemistry and molecular biology gave birth to a protein that was encoded by a host gene, and not by a foreign invader . The concept of a conventional virus was dealt a body blow.
All that we have since learned from molecular biology has added to the presumption of a self-replicating protein as the core or even sole constituent of the infectious agent, and further support has come from an unexpected source - the discipline of epidemiology, at the farthest possible reach in terms of scale from the study of molecules. Anchored to the criterion of experimental transmissibility, the diagnosis of sporadic CJD was being ever more precisely defined, and accurate surveys of disease occurrence confirmed a combination of rarity and randomness that made the idea of contagion very difficult to entertain . Epidemiology also consipired with molecular genetics to show that the few geographic clusters of CJD all resulted, not from environmental peculiarities, but from familial disease due to a genetic mutation in the PrP-encoding gene on chromosome 20. Thus, both sporadic and familial disease appeared unassociated with any outside influence whatsoever: in the immortal words of Pogo, the American comic strip character, "we have seen the enemy, and they are us".
In the past decade, a quite remarkable amount of further study has been undertaken in many different countries and laboratories in an effort to determine the precise basis of infectivity in transmissible spongiform encephalopathy (TSE), and at the same time find some means to protect both humans and animals from becoming infected. On the first count, there have been spectacular if still unfinished results, whereas on the second count the record is marred by three missed opportunities that instead led to tragedies. Two of them (human growth hormone and dura mater grafts) might have been foreseen; the third (bovine spongiform encephalopathy, or BSE) is best ascribed to plain bad luck.
We have learned that PrP is not distinguished from the universe of proteins by any unique structural features – it is in the lower mid-range in size (35,000 Daltons), has an octapeptide-coding repeat region, two asparagine-linked sugar moities enclosed within a disulfide bridge, and a glycolipid membrane anchor.
Because its primary structure is identical in both normal and disease states, it has been concluded that the molecular basis of disease results from a critical switch in the protein’s mix of 3-dimensional patterns from a predominantly α helix to β sheet configuration, changing from a "floppy" soluble to a "stiff" insoluble amyloid, rather like turning a chiffon curtain into a Venetian blind. This conversion has also been accomplished in vitro, but it has so far proved technically impossible to measure a significant parallel change in infectivity.
Although the only cells that appear to be morphologically and functionally compromised lie within the nervous system, the infectious agent is also present in many visceral organs, depending on the host species, route of infection, and agent strain (BSE, for example, has so far shown a very restricted tissue distribution). The most thoroughly studied forms of TSE are caused by oral infections, particularly in experimental models of scrapie, where the major pathogenic pathway, measured both by infectivity and PrP, first involves the tonsils, intestinal lymphatic tissues, and spleen, from which it spreads via the splanchnic nerve into the spinal cord and on up to the brain. A minor alternative route short-circuits the spleen and probably reaches the brain via the vagus nerve. Very recently, it has been found in one scrapie model that B-cells in the blood are critical for neuroinvasion to occur, but it is not yet known whether they act as carriers of the infectious agent, or simply facilitate its transfer from other cells into the nervous system.
More than two dozen different point and insert mutations have been identified in the PrP-encoding gene that are responsible for the familial form of CJD, Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). All occur in a Mendelian dominant pattern of inheritance, and all are experimentally transmissible to laboratory animals. Evidentally, these mutations increase to near certainty the likelihood that during a single lifetime the α helix to β sheet protein transformation will occur, and once transformed, the altered protein sets in motion the cascade of molecular events leading to the generation of amyloid with the property of self-replication. This, at least, is the theory.
Molecular genetic manipulation, although not yet feasible in humans, has provided some extraordinarily interesting results in experimental animals. Mice created with a PrP gene containing the equivalent of one of the human mutations (P102L) spontaneously develop a fatal spongiform encephalopathy . Conversely, mice in which the PrP gene has been either made dysfunctional or excised, and thus do not produce PrP, are totally insusceptible to experimental TSE, and perhaps even more surprising, live to old age in perfect health (apart from an altered circadian rhythm), which suggests that the PrP gene is redundant in mice, and thus perhaps also in humans, opening the door to consideration of gene ablation therapy in human TSE .
One might suppose that all of this basic knowledge might have translated into practical solutions for the prevention of disease. One would be deceived. While these basic research studies were going on, three outbreaks of CJD tested our ability to foresee potential problems, and found it wanting. Beginning around the mid-1960’s, a procedure to extract growth hormone from pituitary glands had been sufficiently refined to permit large scale production and distribution to hormone-deficient patients. Glands were obtained from cadavers at autopsy, and were pooled in batches of up to 10,000 for each production run. In 1985, CJD was reported in three US patients, leading to the immediate replacement of native hormone by a recombinant product. Despite this action, CJD has since been responsible for over 130 additional deaths, chiefly in France, Great Britain, and the U.S., after longer and longer incubation periods (up to 30 years) dating from the period when native hormone was used. It is clear that even when the potential risk was appreciated (nearly ten years before the first case of CJD), most of the damage had already been done because of the decades-long "lead time" between peripheral route infections and disease; moreover, screening criteria were not always effective in preventing the inclusion of pituitaries from unsuspected cases of CJD.
Almost coincident with the growth hormone-CJD outbreak, contaminated dura mater grafts were also discovered to have caused iatrogenic disease: since 1988, more than 100 neurosurgical cases have died from CJD, the contamination again resulting from inadequate donor screening criteria, and batch-pooling before or during processing of cadaveric tissue. The lessons learned from these tragedies have prompted much more stringent regulations governing the collection and use of human-sourced biologicals, particularly those originating from central nervous system tissues.
Scrapie, meanwhile, had been quietly biding its time, waiting for the moment when, through human carelessness or lack of foresight, it would again attain the front ranks of medical attention. That moment came in 1996, with the recognition in British young people of a "new variant" of CJD (nvCJD, or the Will-Ironside syndrome) that has since with near certainty been traced to the consumption of tissue from cattle infected with spongiform encephalopathy (BSE), they having in turn consumed meat and bone meal contaminated with scrapie-infected rendered sheep carcasses . The story is too well known to be rehearsed in detail, and contains elements that are still disputed, but it appears most likely that changes in the animal rendering process that occurred around 1980 allowed the scrapie agent to survive and infect cattle, the carcasses of which were then recycled through the rendering plants, leading to ever greater levels of cattle-adapted infectivity in meat and bone meal, and eventually producing a full-scale BSE epidemic.
Recognition of this source of infection led to the imposition in 1988 of a ruminant feed ban that by 1992 had turned the epidemic around, but the loss of some 180,000 cattle to date has brought the British livestock industry to its knees. BSE has also echoed through the tallow, gelatin, and pharmaceutical industries, all of which make use of bovine-derived products for human use, and even the blood-bank community has been seriously affected by virtue of the uncertainty about infectivity in blood donations from patients incubating nvCJD. There are presently just over 50 verified cases of nvCJD, and the number continues to grow at the rate of about 10 new cases per year: whether they represent a small group of susceptible individuals, or the leading edge of a major epidemic is still moot.
Despite these battle scars from engagements in applied science, we can look back with some satisfaction upon the accomplishments in basic science during the century now drawing to a close, and expect that during the early years of the 21st century, most of the remaining uncertainties will be resolved. These can be grouped into four broad categories: precise characterization of the infectious agent, elucidation of the mechanism of agent replication; prevention or treatment of disease; and continued exploration for other candidate diseases.
Although PrP is beyond doubt a necessary component of the infectious agent, and a growing body of evidence points to the likelihood that it is not only necessary but sufficient to be considered as the infectious agent, formal proof is still lacking. This may come from continuing attempts to demonstrate a parallelism between the amount of infectivity and the in vitro conversion from normal to abnormal protein isoforms, or from the creation of infectious synthetic PrP polypeptide sequences, or from the native protein processed to crystalline purity, guaranteed free from any contaminating molecular species, yet still able to transmit disease.
Precise characterization of the infectious agent will not by itself solve the question of PrP "replication". What is it about PrP amyloid (as distinct from other types of amyloid) that gives it the ability to replicate and transmit disease to new hosts? We know that the β A4 amyloid of Alzheimer’s disease also derives from a normal host protein that in diseased individuals accumulates in the brain, but it does not have the ability to transmit disease to a healthy individual. Why this difference?
The answer to this fundamental biological question need not inhibit research into disease prevention and therapy, which may come from a more general understanding of the process of amyloid formation. Chemical manipulation of the cellular pathways involved in PrP metabolism, or interference with the α helix to β sheet configura- tional shift to amyloid (the CNS version of a "beta-blocker") could become viable therapeutic approaches, and efforts to arrest and even reverse amyloid accumulation in experimental models are already beginning to show promise. Similarly, manipulation of the PrP gene (or its expression) in familial forms of disease will become feasible when genetic engineers overcome the technical problems that have generally prevented the successful results obtained in mice to be duplicated in humans.
Finally, we must continue to keep alert to the possibility that other diseases without known cause may share the apparently unique pathogenic mechanism of TSE, and so be susceptible to the same therapeutic approaches. We should also be prepared to admit that however interesting as a biological phenomenon, "replicating proteins" may not be found to cause other more numerically important disorders, but like Rickettsiae (with equal claim to biological uniqueness, and causing only four uncommon diseases), may forever remain confined to the small group of presently recognized "prion diseases" that pose a comparatively minor burden to human health.
Paul Brown, MD
Raymond Bradley, DMV
N.B. This essay is a slightly modified version of a paper that appeared in the British Medical Journal in the final issue of 1998 (Dec 19-25)
Journal of the House of Commons, 1755, vol 27, p 87.
Leopoldt JG. Nützliche und auf die Erfahrung Gegründete. Einleitung zu der landwirthschaft, funf Theile. Christian Friedrich Günthern, Berlin and Glogau, 1759, p 348.
Besnoit C, Morel C. Note sur les lésions nerveuses de la tremblante du mouton. Rev Vet 1898; 23: 397-400.
Besnoit C. La tremblante ou névrite périphérique enzootique du mouton.
VI. Etiologie. Rev Vét 1899; 23: 307-43.
Cuillé J, Chelle P-L. La maladie dite "tremblante" du mouton est-elle inoculable? Compte Rend Acad Sci 1936; 203: 1552.
Gordon WS. Advances in veterinary research. Vet Rec 1946; 58: 516-20.
Dickinson AG, Fraser H. An assessment of the genetics of scrapie in sheep
and mice. In: Slow Transmissible Diseases of the Nervous System, vol 1, Prusiner SB, Hadlow WJ, eds, Academic Press, New York, pp 367-86.
Alper T, Cramp WA, Haig DA, Clarke MC. Does the agent of scrapie replicate without nucleic acid? Nature 1967; 214: 764-6.
Chandler RL. Encephalopathy in mice produced with scrapie brain material. Lancet 1961; i: 1378-9.
Hadlow WJ. Scrapie and kuru. Lancet 1959; ii: 289-90.
Gajdusek DC, Zigas V. Degenerative disease of the central nervous system in New Guinea: epidemic occurrence of "kuru" in the native population. N Engl J Med 1957; 257: 974-8.
Gajdusek DC, Gibbs CJ Jr, Alpers M. Experimental Transmission of a kuru-like syndrome to chimpanzees. Nature 1966; 209: 794-6.
Klatzo I, Gajdusek DC, Zigas V. Pathology of kuru. Lab Invest 1959: 8: 799-847.
Gibbs CJ Jr, Gajdusek DC, Asher DM, Alpers MP, Beck E, Daniel PM,
Matthews WB. Creutzfeldt-Jakob disease (spongiform encephalopathy): transmission to the chimpanzee. Science 1968; 161: 388-9.
Bolton DC, McKinley MP, Prusiner SB. Identification of a protein that purifies with the scrapie prion. Science 1982; 218: 1309-11.
Oesch B, Westaway D, Wälchli M, McKinley MP, Kent, SBH, Aebersold R, et al. A cellular gene encodes scrapie PrP 27-30 protein. Cell 1985; 40: 735-46.
Hsiao K, Scott M, Foster D, Groth DF, DeArmond SJ, Prusiner SB. Spontaneous neurodegeneration in transgenic mice with mutant prion protein. Science 1990; 250: 1587-90.
Büeler H, Fischer M, Lang Y, Bluethmann H, Lipp H-P, DeArmond J, et al.
Normal development and behaviour of mice lacking the neuronal cell-surface
PrP protein. Nature 1992; 356: 577-82.
Brown P, Cathala F, Raubertas RF, Gajdusek DC, Castaigne P. The epidemiology of Creutzfeldt-Jakob disease: conclusion of a 15-year investigation in France and review of the world literature. Neurology 1987; 37: 895-904.
Will RG, Ironside JW, Zeidler M, Cousens SN, Estibeiro K, Alperovitch A, et al. A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 1996; 347: 921-5.