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,~ Prion Diseases: An Overview This chapter provides the historical backdrop for today's research into prion diseases, also called transmissible spongiform encephalo- pathies (TSEs). Most of this history occurred during the past cen- tury. In fact, the outbreaks of bovine spongiform encephalopathy (BSE) and variant Creutzfel~t-Takob disease (vCTD) catalysts for much of the present research on TSEs occurred within the past two decades. Prions, also called PrPSc, and their normal, cellular isoform, PrPC, are encoded by the PRNP gene on chromosome 20 in humans. Like all pro- teins, prpC has a characteristic conformation. Under certain conditions, however, it folds into an abnormal shape that is associated with fatal neurodegeneration after a long incubation period. This report uses the terms prion and PrPSc interchangeably in reference to the abnormally folded, pro- tease-resistant protein associated with TSEs. However, the committee is sensitive to the fact that such usage is controversial. We want to explain our choice of words in the context of this controversy. If prion aggregates represent the infectious agent that causes TSEs and if prions also are the misfolded protein known as PrPSc, then by parallel reasoning, aggregates of PrPSc represent the infectious agent of TSEs. This view, known as the protein-only theory, has many proponents. Yet some reputable TSE experts believe that PrPSc alone may be insufficient to cause a TSE infection, although the protein may be associated with and even neces- sary for such an infection. A number of these investigators hypothesize that the infectious particle may contain hidden nucleic acid, additional proteins, or some other additional, essential material. This camp uses the term prion to refer to TSE infectivity, but does not equate prion with PrPSc. To respect 39
40 ADVANCING PRION SCIENCE and recognize this alternative point of view, the report often uses the phrase "the infectious agent of ELSE under discussion]" instead of the term prion or PrPSc. The committee believes that the preponderance of scientific evidence favors the hypothesis that priors, consisting of PrPSc, are associated with infection in TSEs. Nevertheless, the purpose of this report is not to resolve the controversy or to proffer the committee's opinion regarding the prion hypothesis. Rather, the purpose of this report is to call for additional re- search especially studies of the fundamental molecular structures and mechanisms related to TSEs so that disparate views may converge and ac Vance prlon science. ORIGINS AND DEVELOPMENT OF PRION SCIENCE The identification of a previously unknown malady in the Fore Tribe of Papua New Guinea drew international attention to the group of brain-wast- ing diseases called TSEs. Physicians Vincent Zigas and Daniel Carleton Gaj~usek in 1957 described an epidemic among the Fore people character- ized by loss of balance, dementia, and death (Gaj~usek and Zigas, 19571. The tribe called the illness kuru, meaning to tremble or to shiver. Studies of the brains of deceased patients revealed widespread neurodegeneration marked by vacuoles in the cytoplasms of nerve cells (Klatzo et al., 19591. The vacuoles gave the victims' brains a spongelike appearance at the micro- scopic level hence the term spongiform encephalopathy. Veterinary neuropathologist William Hadiow was the first to recognize similarities between kuru and scrapie, a TSE of sheep and goats that had been known since the 1700s. He pointed out in a 1959 letter to The Lancet that the brains of mammals with both conditions had a unique form of widespread neuronal degeneration. "Large single or multilocular 'soap- bubble' vacuoles in the cytoplasm of nerve-cells have long been regarded as a characteristic finding in scrapie," he wrote; "this extremely unusual change, apparently seldom seen in human neuropathological material, also occurs in kuru, and first aroused my curiosity about the possible similarity of the two diseases" (Hadiow, 1959:2901. Scrapie and kuru both were endemic to specific populations in which the usual incidence was low, he added. Clinical symptoms could appear months after a victim had been separated from the source community, and both diseases were found in previously healthy communities after the intro- duction of an individual from a known source community. Hadiow also noted that data suggested a genetic predisposition toward both diseases, which did not appear to be infectious in the traditional sense. Victims ex- hibited increasingly severe ataxia, tremors, and behavioral changes, yet no consistent abnormalities appeared in their blood or cerebrospinal fluid. Both
PRION DISEASES: AN O VER VIE ~ diseases began insidiously, he wrote, "and usually end fatally 41 . . .; only rarely have remissions and recoveries been observed") (Hadiow, 1959:290~. On the basis of these observations, Hadiow suggested that experimen- tal transmission of kuru into nonhuman primates might prove fruitful, since veterinary scientists were successfully investigating scrapie by inoculating healthy sheep and goats with brain tissue from animals with the disease. After extensive work, Gaj~usek and colleagues did transmit a "kuru-like syndrome" with an incubation period of 18 to 21 months to chimpanzees by inoculating them with brain suspensions from kuru patients (Gaj~usek et al., 1966), indicating that a noninflammatory neurodegenerative disease could be transmissible. Ethnological and epidemiological studies indicated that kuru was trans- mitted during an endocannibalistic2 funeral ritual (Alpers, 1968; Gaj~usek, 1977; Glasse, 1967~. Women would remove the brain of a deceased rela- tive, eat it along with other tissues, and smear it over their bodies and those of young children of both sexes (Gaj~usek, 1977; Glasse, 1967~. Women who fell victim to kuru outnumbered men who fell victim to the disease by more than 14 to 1 (Gaj~usek and Zigas, 1957~. After a 1957 ban on canni- balism in Papua New Guinea, the number of kuru cases gradually declined over decades, reaching single figures in recent years (Huillard d'Aignaux et al., 2002; Klitzman et al., 1984~. Similarities in the neuropathology of kuru and of a rare, fatal condition called Creutzfel~t-Takob disease (CTD) led investigators to attempt experi- mental transmission of CTD to nonhuman primates. The disease was trans- mitted successfully to a chimpanzee, which first displayed clinical signs af- ter a 1 3-month incubation period, providing more evidence that spongiform encephalopathies are transmissible (Gibbs et al., 1968~. These studies and observations generated a groundswell of interest in discovering the nature of the infectious agent or agents that caused scrapie and kuru. Although many scientific articles referred to the scrapie agent as a slow virus, a number of new hypotheses on the nature of the kuru and scrapie agents surfaced between 1962 and 1981, ranging from a small DNA virus to a replicating polysaccharide to naked nucleic acid similar to plant viroids (Prusiner, 1982~. None of these explanations gained widespread ac- ceptance, however, and the cause of scrapie remained an enigma. iIt was later discovered that the apparent cases of kuru reported to be in remission or recovery were not, in fact, kuru. In other words, kuru is uniformly fatal. 2Endocannibalism: humans eating the tissue of other humans who belong to their tribe.
42 ADVANCING PRION SCIENCE The Birth of Molecular Prion Science A rapid succession of discoveries about scrapie during the early to mid- 1980s marked the birth of molecular prion science. In 1981, scientists first recognized the rod-shaped structures named scrapie-associated fibrils in concentrated suspensions from scrapie brain (Mertz et al., 1981~. Investiga- tors identified in partially purified samples of such suspensions the pre- dominant protein band that proved to be the prion protein (PrP) (Prusiner et al., 1981~. In 1982, Prusiner asserted that the infectious agent of scrapie was either a protein or a small nucleic acid surrounded by a tightly packed protein (Prusiner, 1982, 1999~. He called this infectious agent a prion, which stands for "small, proteinaceous infectious particles that are resistant to inactiva- tion by most procedures that modify nucleic acids" (Prusiner, 1982:141~. At the time, the theory that replication of microorganisms and viruses re- quires nucleic acids was well established. Yet several investigators had iconoclastically proposed that the infectious agent of scrapie might not re- quire nucleic acids and could be a replicating protein (Alper et al., 1967; Griffith 1967; Lewin, 1972; Pattison and Tones, 1967~. Until Prusiner's en- try into the field, however, no investigator had provided compelling data to support this hypothesis. Applying advanced biochemical techniques, Prusiner demonstrated that the scrapie agent resisted six different procedures known to attack nucleic acids and was susceptible to six methods of protein inactivation (Prusiner et al., 1981; Prusiner, 1982~. It was later established that prions and PrPSc were resistant to limited digestion by one of those methods, exposure to proteinase K (McKinley et al., 1983~. Like some investigators whose theo- retical work preceded him, Prusiner (1982:139) correctly suggested that a prion might act as "an inducer or template for its own synthesis." The following year, Diringer and colleagues demonstrated TSE infectivity in fibril-containing suspensions from scrapie-infected brain tissue (Diringer et al., 1983~. The determination of a partial amino acid sequence for PrP (Prusiner et al., 1984) facilitated the production of corresponding oligonucleotides, which were used independently by three groups those of Robakis, Chesebro, and Weissmann to isolate cDNA clones corresponding to the PrP mRNA in scrapie brain tissue (Chesebro et al., 1985; Locht et al., 1986; Oesch et al., 1985; Robakis et al., 1986~. The cDNA clones corresponding to the full-length PrP protein sequence for mouse and hamster were found by the Chesebro and Robakis groups, respectively (Lochs et al., 1986; Robakis et al., 1986~. All three groups found PrP mRNA in both scrapie- infected and uninfected brain, indicating that Prnp was a normal host gene and did not come from the genome of an exogenous infectious agent. ~ 1
PRION DISEASES: AN O VER VIE ~ 43 In addition, Oesch and colleages (1985) reported that PrP from scrapie brain is partially resistant to digestion by proteases, whereas PrP from normal brain is not resistant. This pivotal finding is the basis for the Western blot diagnostic test for the presence of PrPSc in brain and other tissues in virtually all types of human and nonhuman TSEs, as discussed in Chapter 4. After two decades of research, many but not all TSE experts accept the protein-only hypothesis. Prusiner won the Nobel Prize in physiology or medicine in 1997 for his groundbreaking work. But because Koch's postu- lates3 have not been demonstrated for priors, some scientists believe that prions alone do not explain all aspects of the etiology of TSEs (Chesebro, 1998, 1999; Rohwer, 19911. An overview of all known TSEs appears in Table 2-1. THE NATURE OF PRIONS AND PRION PROTEIN The normal, cellular prion protein, PrPC, resides on the membranes of many avian and mammalian cells. Its physiologic function is poorly under- stood, although its function or loss of function could possibly contribute to some aspects of the disease state in TSEs. Primary Structure4 of Human prpC Human PrPC is a string of 253 amino acids (see Figure 2-11. At one end, called the C-terminus, the molecule is attached to the cell membrane by a glycosy! phosphatidylinosito! (GPI) moiety known as the GPI anchor. This anchor is added to the molecule when amino acids 231-253, known as the GPI signal sequence, are removed during a process called transamidation. Two cystine amino acid residues form a tight bond in the C-terminus re- gion. In addition, various complex carbohydrates attach to asparagine amino acids in this region. Some important determinants of the protein's tertiary structure lie in the central portion of the molecule, where the pro- tease-resistant segment resides. Gene mutations or polymorphisms on the PrP gene cause amino acid substitutions on prpC that can either predispose a person to or protect one from TSEs. 3Koch's postulates are "criteria for proving that a specific type of microorganism causes a specific disease. 1 ) The organism should be constantly present in the animal suffering from the disease and should not be present in healthy individuals. 2) The organism must be cultivated in pure culture away from the animal body. 3) Such a culture, when inoculated into susceptible animals, should initiate the characteristic disease symptoms. 4) The organism should be reisolated from these experimental animals and cultured again in the laboratory, after which it should still be the same as the original organism" (Brock et al., 1994:19). 4Primary structure denotes the order of amino acids in a polypeptide.
44 TABLE 2-1 Classification of TSEs ADVANCING PRION SCIENCE Type of TSE Modes of Affected Natural Date First Mammals Transmission Recognized In humans Sporadic Creutzieldt- Jakob disease (sCJD) Unknown 1920 Sporadic fatal insomnia Unknown 1997a (sFI) Familial Creutzieldt- Genetic 1924b Jakob disease Fatal familial insomnia Genetic 1986C (FFI) Gerstmann-Straussler- Genetic 19363 Scheinker disease Kuru Iatrogenic Creutzieldt- Jakob disease Variant Creutzieldt- Jakob disease (vCJD) in animals S. craple Transmissible mink Mink encephalopathy Exposure to 1957 contaminated human . , . tissues curing en do canni b alistic rituals CJD-infected surgical 1974e equipment or tissue transplants Food-borne exposure 1996 to BSE-infected tissue; other modest Sheep, goats Contact with infected 18th sheep, placenta, or century contaminated environment; possible oral exposure Food-borne exposure 1947 to infected tissue Chronic wasting Deer, elk Unknown; likely oral 1967 disease (CWD) Bovine spongiform Cattle encephalopathy (BSE) Food-borne exposure 1986 to TSE-infected tissue
PRION DISEASES: AN O VER VIE ~ TABLE 2-1 Continued 45 Type of TSE Affected Mammals Modes of Natural ~ ransm~ss~on Date First Recognized In animals (continued) BSE (continued) Feline spongiform encephalopathy Nyala, gemsbok, Arabian oryx, eland, kudu, scimtar-horned oryx, puma, cheetah, ocelot, tiger, African lion, Asiatic golden cats Food-borne exposure 1986 to BSE-infected tissue Domestic cat Food-borne exposure 1990 to BSE-infected tissues aMastrianni et al. (1997). bGambetti et al. (1999). This chapter cites Kirschbaum (1924) as reporting the first authentic case of familial Creutzieldt-Jakob disease. CLugaresi et al. (1986). ~Kretzschmar et al. (1991). eDuffy et al. (1974) lit is unknown whether the disease is transmissible by transfusion or transplantation. Ache TSE affecting these zoo animals was called "exotic ungulate encephalopathy" until transmission studies demonstrated that some of the animals had BSE. It is believed that these animals became infected by eating feed or, in the case of exotic feline species, fresh bovine tissues contaminated with the BSE agent. hDomestic cats develop feline spongiform encephalopathy in nature by eating feed contaminated with the BSE agent. SOURCES: Godon and Honstead (1998),Johnson and Gibbs (1998), Haywood (1997); personal communication, E. Williams, University of Wyoming, December 2002; Prusiner (1995), and Young and Slocombe (2003). At the opposite end of the protein, called the N-terminus, lie five re- peating sequences of eight amino acids. It is thought that these "octapeptide repeats" have a strong affinity to copper, giving the protein an antioxidant function (Brown and Sassoon, 20021. The 22 amino acids at the N-terminus are removed as the protein is synthesized in the endoplasmic reticulum. Then the carbohydrates and the GPI anchor are added to the protein, and it migrates to the cell surface membrane. Conversion of prpC to PrPSc Mature human prpC polypeptide consists of amino acids 23-230. The three-dimensional structure of this protein is depicted in Plate 2-1 (Zahn et
46 N-terminus ADVANCING PRION SCIENCE Mature PrP - Unmodified PrP Octapeptide: 129 MN I S-S I repeats ~ 4, l l 1 1 23 51 ~ 91 `' T 1 231 1 253 Signal peptide ~ ~ CHO CHO GPI signalsequence ,' 129 MN ~ / \ / \ / ~ ~ \ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 58 81 90 PrP 27-30 / I \ 146 160 150 C-terminus FIGURE 2-1 Diagram of the primary structure of normal human prion protein, PrPC. The complete, unmodified polypeptide has 253 amino acid residues, while the mature polypeptide extends from residues 23 to 230. An arrow indicates the approximate position of the methionine-valine (MIV) polymorphism at codon 129. Other important features include the two p-sheet regions All, three oc-helical regions (H), internal disulfide bond (S-S), two glycosylation sites (CHO), and sequence of eight residues repeated five times in a row (octapeptide repeats). The lower, enlarged image depicts PrP 27-30, a PrP region of variable length that resists proteinase-K digestion more than any other part of the protein. Vertical dotted lines labeled with the number of an amino acid residue indicate the points at which proteinase K is believed to cleave PrP to produce PrP 27-30. NOTE: GPI = glycosyl phosphatidylinositol. SOURCE: Adapted from Gambetti et al. (1999:512~. al., 20001; notice the three oc-helices and the two flat regions, called p- sheets. By a poorly understood mechanism, prions convert prpC into the abnormally folded conformation (see Plate 2-2), which contains more p- sheets than the normal isoform. In a demonstration of the pivotal role of normal prion proteins in the progression of TSEs, mice in which prpC ex- pression was knocked out or ablated remained healthy after infection with prions (Bueler et al., 19931. It is widely hypothesized that one or more so- called chaperone molecules may play a role in the conversion of prpC to PrPSc (Chernoff et al., 1995; Telling et al., 19951. Figure 2-2 depicts the presumed processes of PrP production, conversion, and degradation (Caughey, 20021. Unlike PrPC, the aberrantly folded PrPSc can aggregate and become in-
PRION DISEASES: AN O VER VIE ~ 47 FIGURE 2-2 Model of PrPSc formation and deposition in a neuron infected with the agent of TSE. Normal cellular prion protein, prPc (grey circles), is produced in the endoplasmic reticulum (ER), processed in the Golgi apparatus, and transported to the cell surface. There, prPc normally has a short half-life as a result of endocytosis followed by proteolytic degradation within lysosomes. In cells infected with the TSE agent, prPc is converted to PrPSc (dark rectangles) on the cell surface or in endosomes. The conversion of prPc occurs upon contact with PrPSc clusters and, perhaps, accessory molecules. Unlike PrPC, PrPSc is resistant to proteolytic degradation and can accumulate within lysosomes, on the cell surface, or in extracellular deposits, such as rod-shaped fibrils or amyloid plaques. These abnormal accumulations are ultimately neurotoxic. However, it is unclear whether the accumulations cause neurotoxicity directly or whether they cause it indirectly through changes induced in accessory cells, such as microglia (which often surround amyloid plaques) or astrocytes (not shown). Reprinted from Caughey and Chesebro (2002) with permission from ASM Press. Copyright 2002 by ASM Press. soluble, in which case it resists complete digestion by proteinase K (Prusiner, 20011.5 To date it appears that the immune system does not recognize and 5PrPSC demonstrates a gradient of resistance to proteinase K (PK) and is associated with infectious potential and TSEs even in circumstances when it is sensitive to PK digestion. By contrast, the term PrPreS stands for abnormally folded prion protein that is highly resistant to PK digestion and is strongly associated with TSEs. PrPreS is sometimes used synonymously with PrPSc, creating confusion about the differences between the two proteins.
48 ADVANCING PRION SCIENCE destroy PrPSc despite its distinct conformation, presumably because it has the same primary structure as PrPC. Pathogenesis of TSEs In experiments in which animals received PrPSc by peritoneal inocula- tion or through the gastrointestinal tract, the proteins migrated to the lymphoreticular system and propagated there (Brown et al., 1999; Weissmann et al., 2001~. The propagation of prions in the lymphoreticular system is not essential for neuroinvasion, however. PrPSc migrates to the brain along peripheral nerves (Beekes et al., 1998; Kimberlin et al., 1983; Oldstone et al., 2002; Race et al., 2000~. The misfolded proteins aggregate into rod-shaped fibrils, and it has been hypothesized that prions at the ends of the rods continue converting normal PrP into PrPSc (Caughey, 2002~. By an unknown mechanism, the aggregated prions appear to destroy nerve cells and create microscopic vacuoles in the brain. Clinically, this destruc- tion manifests itself differently in different species and in different TSEs within the same species, but the process appears to lead inevitably to death. Prion Strains Both the incubation period of a TSE and the length of time between the onset of clinical symptoms and death vary widely depending on the host species and the strain of PrPSc. Investigators initially differentiated among prion strains through clinical observation of goats that displayed either drowsy or hyperactive behaviors (Pattison and Milison, 1961~. Later work with mice revealed that genetic factors play a role in determining strain differences. Dickinson and colleagues identified in inbred mice two differ- ent alleles, called sine (strain incubation) genes, that consistently resulted in a long or short incubation period prior to the onset of disease (1968~. The investigators later published additional findings that strains could be differ- entiated by the distribution of microscopic lesions (vacuoles) in the brain (Fraser and Dickinson, 1973~. Studies using the agent of transmissible mink encephalopathy in hamsters showed differences in clinical presentation and PrPSc glycoform patterns by prion strain (Bessen and Marsh, 1992~. Recently, the use of selected inbred and transgenic mice to characterize prion strains has led to important insights, including the idea that a similar strain causes both BSE and vCTD (Bruce et al., 1997; Scott et al., 19991. Even more recently, physicochemical methods have been employed to char- acterize and differentiate strains. These include the Western blot to demon- strafe different glycoform patterns of PrPSc (Collinge et al., 1996) and the immunoassay to identify different molecular conformations of PrPSc (Safer et al., 19981.
PRION DISEASES: AN O VER VIE ~ 49 Despite these advances, much about prion strains remains a mystery. Researchers must be able to differentiate one strain from another and to determine the scope of strain diversity, the special characteristics of a strain's conformation that make the strain unique, a host's susceptibility to various strains, and how incubation periods and patterns of disease expression vary by strain and host. THE EPIDEMIC OF BSE AND THE EMERGENCE OF vC3D Prion diseases remained obscure outside the circles of infectious disease specialists and neurologists until the mad cow epidemic struck in the United Kingdom. The illness was first recognized in 1985 when a handful of cattle from disparate locations in the United Kingdom began dying of a strange illness marked by insidious onset, progressive behavioral and locomotive signs, and death (Welis et al., 1987; Wilesmith et al., 1988~. Neuropatho- logical examination of the sick cattle's brains revealed abnormal, micro- scopic vacuoles and fibrils, much like the spongiform characteristics of scrapie and kuru. A team of veterinary scientists at the United Kingdom's Ministry of Agriculture reported in 1987 that the new disease strongly re- sembled the so-called unconventional viral-agent encephalopathies previ- ously observed in sheep and the Fore people, so the scientists named the disease bovine spongiform encephalopathy (Welis et al., 1987~. The out- break appeared mainly in dairy cows rather than beef cattle (Anderson et al., 1996) because it was much more common to feed meat-and-bone meal, the attributed source for BSE transmission, to dairy cows than to beef cattle. BSE quickly ballooned into an epidemic that peaked at more than 37,000 annual cases in the United Kingdom in 1992 (see Figure 2-3) (De- partment for Environment, Food and Rural Affairs, 2002~. It has been esti- mated that 840,000 to 1.25 million infected animals entered the human food chain from 1974 through 1995 (Anderson et al., 1996; Wilesmith et al., 1992~. Conscious of the fact that the transmission of BSE to humans was a possibility, the United Kingdom in 1990 increased epidemiological surveil- lance of CTD, a rare human spongiform encephalopathy. It was thought that changes in the pattern of CTD could signify a link to BSE. The neuro- pathological profiles and age distribution of 10 of the 207 patients with CJD examined between 1990 and 1997 differed markedly from those typi- cal for CTD (Will et al., 1996~. This discovery led to the conclusion that a new variant of CTD had arisen in the United Kingdom. Biochemical studies revealed that the new variant, vCTD, involved the same prion strain impli- cated in BSE (Collinge et al., 1996), and transmission studies with inbred mice confirmed this finding (Bruce et al., 1997; Collinge et al., 1996~. There is evidence of genetic susceptibility to vCTD. A 1997 study found
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PRION DISEASES: AN O VER VIE ~ 51 that a specific genotype of codon 129 in PRNP was correlated with vCTD in all 26 patients tested (Zeidler et al., 1997~. This codon normally codes for methionine on one allele and valine on the other, or the alleles may be homozygous, but the PRNP genes from all patients tested were consistently homozygous for methionine (Collinge et al., 1996~. It is possible, however, that individuals with the other genotypes are susceptible, but that their in- cubation periods may be longer. It is well known that substantial variation in the incubation periods of strains of a mouse-adapted scrapie agent results from their passage through mice with different PrP genotypes. As of September 3,2003,136 residents of the United Kingdom have died of definite or probable vCTD since 1990 (The UK Creutzfel~t-Takob Disease Surveillance Unit, 2003~. Until recently, graphs of the quarterly incidence of vCTD onsets and deaths took an exponential trajectory, sug- gesting the epidemic would grow for the foreseeable future. Then in early 2003, scientists reported for the first time that the quarterly incidence of vCTD onsets and deaths in the United Kingdom no longer appeared to be increasing exponentially (see Figure 2-4) (Andrews et al., 2003~. This find- ing suggested that the primary epidemic,6 which includes exclusively indi- viduals who are homozygous for methionine at PRNP codon 129, might ultimately be less extensive than expected. Additional evidence for a smaller-than-expected primary epidemic comes from Ghani and colleagues (2003~. After analyzing the United Kingdom's vCTD statistics through 2002, the group forecast that the worst- case number of PRNP 129 methionine-homozygous vCTD cases will be lower than the group had previously projected. According to Andrews and colleagues (2003:751) it would be prema- ture to conclude that the overall vCTD epidemic is in permanent decline. They refrain from predicting the epidemic's size or end point because the new statistical trend a quadratic mode! is appropriate only for short- term forecasts (2003~. Moreover, they note, the epidemic's future is clouded by many unknowns: the uncertain incubation period of vCTD in people who are heterozygous at PRNP codon 129 or are homozygous for valine; the possibility that subgroups within the methionine-homozygous popula- tion have different incubation periods; the possibility that there are uniden- tified strains of BSE that incubate longer in humans than does the known strain; and the possibility of human-to-human transmission through blood products, surgical instruments, or tissue transplants. 6Scientists speculate that a secondary vCJD epidemic may occur in the future in individuals who are homozygous for valine (V/V) or are heterozygous (M/V) at PRNP codon 129 because they may incubate the disease longer than the methionine-homozygous population.
52 10 8 SO 6 A 4 .O o ADVANCING PRION SCIENCE 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2/94 4/94 2/95 4/95 2/96 4/96 2/97 4/97 2/98 4/98 2/99 4/99 2/00 4/00 2/01 4/01 2/02 4/02 Quarter/Year ~ ~ ~ ~ ~ ~ Fitted quadratic trend - - 95 percent confidence limits Observed quarterly incidence of vCJD onsets (United Kingdom) o Data point for observed quarterly incidence - - -x- - - Expected quarterly incidence FIGURE 2-4 The best-fit curve for the observed quarterly incidence of vCTD onsets in the United Kingdom through December 2002 is quadratic. The fitted quadratic trend appears within its 95 percent confidence limits and has been adjusted for the time delay between onset and diagnosis. SOURCE: Will (2003). GLOBAL IMPACT OF BSE AND vC3D The BSE outbreak and fear of vCTD, coupled with the outbreak of another animal illness, foot-and-mouth disease, devastated the United Kingdom's beef industry. Hundreds of thousands of cattle have been slaughtered as suspect cases or culled because of BSE. Many other coun- tries, especially in Europe, unknowingly imported BSE-positive cattle, beef products, meat-and-bone meal, and ruminant feed from the United King- dom before the BSE epidemic and its cause had become apparent. Conse- quently, these countries also have suffered outbreaks of BSE, culling of herds, public panic, financial losses, and political repercussions. In addi- tion, the United Kingdom's trading partners have banned the import of cattle, beef products, meat-and-bone meal, and ruminant feed from that country. Similar import bans have been imposed on other countries most
PRION DISEASES: AN O VER VIE ~ 53 recently Canada7 that have reported or are at risk for BSE, as discussed in Chapter 7. Two recent reports (GAO, 2002; Harvard Center for Risk Analysis and Tuskegee University Center for Computational Epidemiology, 2001) sug- gest that the U.S. government should strengthen its policies designed to avert BSE and vC]D, although the first of these studies concludes that the risks of a BSE outbreak in the United States are minimal. Chapters 6 and 7 examine these reports in the context of U.S. surveillance for and prevention of TSEs. Chapter 7 also reviews an analysis of the potential impact of a single case of BSE in the United States (Matthews and Perry, 2003) and examines the repercussions of the BSE-positive cow discovered in Canada in May 2003. THE SPREAD OF CHRONIC WASTING DISEASE IN THE UNITED STATES As noted earlier, although the BSE epidemic that struck Europe has spared the United States thus far, chronic wasting disease (CWD) is affect- ing free-ranging and captive deer and elk in several midwestern and western states; it has also occurred in Canada. The unusual, insidious, and fatal illness began appearing in a captive herd of mule deer in the late 1960s at a research facility in Fort Collins, Colorado (Williams and Young, 1980~. The disease affected young adult deer that had been captive for approxi- mately 2.5 to 4 years. Sick animals became listless, depressed, and anorexic; they died of emaciation, secondary complications, or euthanasia within 2 weeks to 8 months after the onset of clinical signs. The nature of these signs led biologists to name the illness chronic wasting disease. The most striking and consistent pathological features observed by the early CWD researchers were nerve cell degeneration and widespread micro- scopic vacuoles in the neurons of the brain and spinal cord trademarks of the spongiform encephalopathies previously described in sheep, goats, and humans. This commonality led scientists Elizabeth Williams and Stuart Young to conclude in 1978 that CWD was a new spongiform encephalopa- thy. Captive Rocky Mountain elk living in the same Colorado and Wyo- ming facilities as the affected deer were diagnosed with the disease a few years later (Williams and Young, 19 82 ). Unlike BSE, CWD can spread efficiently from an infected animal to an uninfected animal of the same species, as well as to related species, either directly after exposure or indirectly from the pasture occupied by an in- 7EDITORS' NOTE: After this report was completed, the first U.S. case of BSE was identi- fied in Washington State and was announced to the public on December 23, 2003.
54 ADVANCING PRION SCIENCE fected animal (Gross and Miller, 2001; Williams and Miller, 2002~. It is clear that the disease can be transmitted among mule deer, white-tailed deer, and elk (Williams and Miller, 2002~. The lack of understanding of how CWD spreads and whether it can cause disease in humans and cattle justifies research on the disease, as well as the development of tools for detecting the infectious agent in humans, animals, and the environment. We discuss such tools and research in Chapters 4 and 6, respectively. Major media outlets reported on the growing number of CWD-infected deer and elk in North America during 2002 (Blakeslee, 2002; Regalado, 2002~. Although the disease appears to be spreading, the larger numbers and wider geographic distribution of CWD cases also may reflect more active surveillance during the past several years. This surveillance has re- sulted in the identification of foci of CWD that have existed for a decade or two in the wild and on game farms the news is their discovery (Miller et al., 2000~. Most of the current CWD epidemics in free-ranging and farmed cervids appear to be independent of each other, although they may have a common origin dating back several decades (Williams and Miller, 2002~. It is unknown how the disease initially arose. The spread of CWD among free-ranging cervids will likely follow the animals' predictable, natural movements. Some researchers speculate that CWD in farmed animals has spread more widely and unpredictably as a result of market forces (Williams and Miller, 2002~. UNIQUE CHALLENGES IN CONDUCTING TSE RESEARCH Much about TSEs remains unclear: how prions replicate, why they tar- get neurons, and whether prions or some other entity kill neurons are but a few examples. TSE research has progressed slowly because of a number of challenges unique to the field. First and foremost, prions are an entirely new type of infectious entity, precluding the use of many tools designed for study- ing infectious diseases. Moreover, since PrPSc and prpC have identical amino acid sequences, the existence of a prior-specific antibody has not been con- firmed to date, and infected individuals do not exhibit a prior-specific im- mune response. In addition, prions replicate sluggishly in existing cell cul- ture systems and incubate for several months to several years in animal models, limiting the pace of research. TSE investigators face not only scientific challenges but logistical ones as well. Their work often must take place in laboratories designed for re- search with biohazardous materials; these laboratories are expensive to con- struct, and the United States has few such facilities for prion research. In addition, standardized reagents are difficult to come by. There is only one U.S.-based repository for vCTD tissue, and U.S. scientists need repositories in this country for other reagents and transgenic animals. Until recently, the
PRION DISEASES: AN O VER VIE ~ 55 federal government's limited interest in prion diseases meant that it was relatively difficult to win research grants to study them, and this apparent lack of financial stability has discouraged young scientists from entering the field. Hence, the community of TSE researchers in the United States is small. Sensitive, specific TSE diagnostics would help protect people and ani- mals from fatal prion infections in the absence of prophylactics and treat- ments. Given that there are few tools to inactivate priors, the ability to test blood and other tissues for prions would help prevent the inadvertent trans- mission of vCTD by blood transfusion or organ transplantation, provided that there is actually any infectious agent to be detected in blood or the organs used for transplantation (see Chapter 51. Despite many attempts in Europe and the United States, no one has developed a reliable antemortem diagnostic test for TSEs. The next chapter describes the technologies that offer the greatest promise for achieving this important goal. REFERENCES Alper T. Cramp WA, Haig DA, Clarke MC. 1967. Does the agent of scrapie replicate without nucleic acid? Nature 214(90):764-766. Alpers MP. 1968. Kuru: implications of its transmissibility for the interpretation of its chang- ing epidemiological pattern. In: Bailey OT and Smith DE, editors. The Central Nervous System: Some Experimental Models of Neurological Diseases. Baltimore, MD: Williams and Wilkins. Pp. 234-251. Anderson RM, Donnelly CA, Ferguson NM, Woolhouse ME, Watt CJ, Udy HJ, MaWhinney S. Dunstan SP, Southwood TR, Wilesmith JW, Ryan JB, Hoinville LJ, Hillerton JE, Aus- tin AR, Wells GA. 1996. Transmission dynamics and epidemiology of BSE in British cattle. Nature 382(6594):779-788. Andrews NJ, Farrington CP, Ward HJ, Cousens SN, Smith PG, Molesworth AM, Knight RS, Ironside JW, Will RG. 2003. Deaths from variant Creutzieldt-Jakob disease in the UK. Lancet 361(9359):751-752. Beekes M, McBride PA, Baldauf E.1998. Cerebral targeting indicates vagal spread of infection in hamsters fed with scrapie. Journal of General Virology 79(Pt 3):601-607. Bessen RA, Marsh RF. 1992. Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters. Journal of General Virology 73(Pt 2):329-334. Blakeslee S. 2002, September 3. Brain disease rises in deer, scaring hunters. The New York Times. Science, p. 1. Brock TD, Madigan MT, Martinko JM, Parker J. 1994. Biology of Microorganisms: Seventh Edition. Englewood Cliffs, NJ: Prentice-Hall. Brown DR, Sassoon J. 2002. Copper-dependent functions for the prion protein. Molecular Biotechnology 22(2):165-178. Brown KL, Stewart K, Ritchie DL, Mabbott NA, Williams A, Fraser H. Morrison WI, Bruce ME. 1999. Scrapie replication in lymphoid tissues depends on prion protein-expressing follicular dendritic cells. Nature Medicine 5(11):1308-1312. Bruce ME, Will RG, Ironside JW, McConnell I, Drummond D, Suttie A, McCardle L, Chree A, Hope J. Birkett C, Cousens S. Fraser H. Bostock CJ. 1997. Transmissions to mice indi- cate that "new variant" CJD is caused by the BSE agent. Nature 389(6650):498-501. Bueler H. Aguzzi A, Sailer A, Greiner RA, Autenried P. Aguet M, Weissmann C. 1993. Mice devoid of PrP are resistant to scrapie. Cell 73(7):1339-1347.
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