The subacute spongifrom encephalopathies are associated with unconventional agents whose purification and exact physical identification are yet to be achieved. For many years, scrapie was known to be caused by a transmissible agent where a subacute, steadily progressive and invariably fatal disease is seen. Pathologically, the brain showed areas of gliosis, neuronal loss, vacuolation of neurons, and no inflammatory response. "Scrapie" was used to described the tendency of affected animals to scrape their coats against fences because of pruritus. The disease can be transmitted from animal to animal by parenteral inoculation and could be transmitted to laboratory mice. Scrapie was classified as a "slow infection", akin to other animal diseases such as visna and maedi. It was subsequently noted that scrapie was very similar to Kuru and Creutzfeldt Jacob syndrome, and it was subsequently shown that disease could be transmitted from patients suffering from Kuru and CJD to chimpanzees and other primates. During the 1970s, the major thrust of the research was in purification of the infectious agent.
The following diseases are associated with the unconventional agents:
Creutzfeldt-Jacob (CJD, sporadic and inherited)
Scrapie of sheep and goats
Transmissible mink encephalopathy
Bovine spongiform encephalopathy
scrapie in captive animals
The duration of both kuru and CJD ranges from 6 months to a year and therefore the incidence and prevalence are approximately equal.
Kuru - kuru was restricted to the Fore people in Papua New Guinea who practiced ritualistic cannibalism. The disease is most commonly seen in women, probably because of the fact that the brain and other internal viscera were given to women and children whereas the muscle to men. At the height of the epidemic, over 90% of all deaths in women were due to Kuru. With the cessation of cannibalism in the 1950s, the incidence of Kuru gradually disappeared, first amongst children, and successively thereafter among adolescents and young adults. No one born in a village since cannibalism ceased has ever developed kuru.
CJD - CJD has a worldwide distribution and had been reported in 50 countries. Epidemic clusters have not been documented in the majority of countries affected. However, several foci have been identified in Czechoslovakia, Italy, Hungry and Israel. There is an usually high incidence of CJD in Libyan-born Israeli Jews (more than 30 per million) which is associated with familial clustering. Up to 15% of all cases of CJD have a family history of disease consistent with autosomal dominant transmission. The onset of disease in familial cases is significantly earlier than in sporadic cases. The precise mechanism of spread in familial cases are uncertain. What is known is that the sequence of the PrP gene is changed in affected individuals which may render more susceptible to infection by the agent.
The only demonstrated transmission of CJD has been iatrogenic (contaminated surgical instruments such as EEG electrodes, organ transplants such as cornea transplants, and growth hormone prepared from human pituitaries). It is of some concern that the incidence of previous surgery or other neurological diseases seem to be increased in patients with CJD compared to the normal population. The scrapie agent is closely related to the CJD agent as both causes an identical disease when inoculated into nonhuman primates. However there is no evidence that CJD is caused directly by the scrapie agent, through the consumption of infected sheep. The worldwide distribution of scrapie does not correspond to that of CJD. eg. Australia is free of scrapie but the incidence of CJD is similar to scrapie affected countries.
GSS - GSS is a very rare, usually familial, neurodegenerative disease that affects patients in the third to the seventh decade of life. The exact incidence of GSS is unknown but is estimated to be between 1 to 10 per 100 million. Familial cases are associated with autosomal dominance inheritance in families. A change in codon 102 from proline to leucine was found in the PrP gene of affected individuals. Therefore, it appears this genetic change is essential for the acquisition of the disease.
Scrapie - scrapie is found worldwide except in Australia. In sheep it is thought that both horizontal and vertical transmission can occur. It is not certain how horizontal transmission occurs although cells of the R-E system are infectious.
Transmissible Mink encephalopathy - epidemics have arisen in farmed mink who have been fed sheep offal. It is thought that the scrapie agent is transmitted to farmed mink in this manner. However, in an outbreak in 1985, the mink were fed exclusively on bovine and equine offal, despite the fact that BSE had yet to be reported in the U.S.
BSE - BSE is confined almost exclusively to Britain and
is thought to have arisen as a result of the feeding of "meat
and bone meal", which includes waste from sheep, cattle and
other animals to cattle. The disease is expected to die out once
the ban on the practice of feeding ruminants with the wastage of
ruminants takes effect. This, of course depend on the premise
that no horizontal transmission takes place between cattle.
Kuru is probably transmitted through cannabalism. The gradual decline of the disease once cannabalism had been outlawed provides the strongest evidence. Moreover, there has been no convincing evidece that ct case to case transmission occurs. There are 3 theories as to how cases of CJD occurs;-
1. Cases to case transmission - whether horrizontal, vertical, familial, or iatrogenic through surgical instruments or contaminated drugs.
2. Activation of an endogenous or latent virus infection.
3. Zoonotic transmission of scrapie virus from sheep, goats or other animals.
Of the 3 main hypotheses, the zoonotic transmission of scrapie is the most difficult to test, in spite of the fact that CJD occurs in areas where scrapie is absent. Details of subclinical infection in domestic and wild animals are unknown. (Note the occurrence of an epidemic of transmissible mink encephalopathy in a mink colony in 1985 who have been fed exclusively on cattle and horse waste). In contrast with kuru and CJD, which can be transmitted to chimpanzees and other subhuman primates, scrapie could not be transmitted to chimpanzees although it can be transmitted to other subhuman primates. Activation of an endogenous (latent virus) infection is definitely a possibility in cases of familial CJD and GSS. As far as case to case transmission is concerned, this had been definitively demonstrated in the case of iatrogenic transmission through surgical instruments, organ transplants and human derived growth hormone. Therefore the conclusion is that the natural mechanism of spread and the reservoir of CJD virus remains unknown at the present.
The long incubation period is common to all diseases associated with the unconventional agents. The youngest patient with kuru is 4 years old and died at 5. Conversely, cases of kuru are still seen after the cessation of cannibalism 30 years ago. The incubation period may be related to the dose of the inoculum. Cases of CJD associated with iatrogenic inoculation arose within a 2 year incubation period. However, familial cases of CJD occur commonly in the fourth and fifth decade, suggesting that incubation periods may exceed 4 to 5 decades.
Recently there had been intense speculation on a possible
relationship between CJD and Alzheimer's disease. Alzheimer's
disease shares a number of clinical and pathological features in
common with CJD. Clinically, many patients with Alzheimer's
disease with rapid deterioration developing myoclonus and an
abnormal EEG similar to that seen in CJD. Pathologically, there
is a spectrum of amyloid deposition which overlaps in both
diseases. The most intriguing relationship to emerge has been the
recognition that Alzheimer's disease amyloid is derived from a
glycosylated membrane protein which had been designated as A4.
Therefore it is possible that similar mechanisms may operate in
both Alzheimer's disease and in unconventional virus diseases in
that the host-encoded proteins are rendered amyloidogenic.
CJD presents as a subacute dementia, evolving over weeks to several months and is accompanied by pyramidal, extrapyramidal, and cerebellar signs. The mean age of death is 57 years but the disease may occur in the late teens and early twenties. At various stages of the progression of the disease, myoclonus with periodic sharp-wave complexes in the EEG appear. In the final stages of the disease, there is an incapacitating dementia, usually with severe myoclonus. Patients with GSS present initially with ataxia or dementia and deteriorate inexorably to death over 1 to 10 years. Protease resistant PrP and PrP immunoreactive plaques with characteristic morphology accumulate in the brains of patients with GSS.
Recently, a variant of CJD (vCJD) was described in UK in
France with distinct clinical and pathological features which
affected younger individuals. Behavioral changes occured early in
the course of the illness while EEG changes usually associated
with conventional CJD were not seen. In addition o spongiform
changes, PrP plaques were seen which had an extensive
distribution. The only risk factor common to all these patients
were that all of them had consumed beef. Moreover, the biological
properties of the agent in vCJD and BSE share common biological
properties e.g. they have similar incubation periods in various
kinds of mice and hamsters.
There are no laboratory tests for definitive diagnosis short
of transmission experiments. In the absence of facilities for
these tests, confirmatory diagnosis is made on histopathological
changes in the CNS by light microscopy or EM. The isolation of
scrapie-associated filaments and visualization by EM is also
proving to be of some diagnostic value. By far the majority of
cases of CJD present little diagnositc problem when the
spongiform change is well developed in the cerebral cortex,
striautim, medial thalamus, and the molecular layer of the
There is no known specific treatment for these infections
except symptomatic treatment. Kuru has been virtually eliminated
by the cessation of cannibalism. Iatrogenic transmission of cases
of CJD can be prevented by the proper disinfection of potentially
contaminated materials. There is no epidemiological eveidence
that medical or ancillary personnel are at increased risk of
developing CJD. There is no indication for the isolation or
barrier nursing of a CJD patient. Medical laboratory technicians
should take the same care as that already exercised in clinical
specimens potentially infectious for hepatitis B and HIV,
although to date, no confirmed cases of laboratory-acquired cases
of CJD are known.
Properties of the Agents
Scrapie was the first of this group of agents to be adapted to serial passage in mice and other small rodents, which facilitated easier study because of the shorter incubation periods in these animals. Most of the knowledge of the physicochemical and biological properties of these agents had been derived from studies in mice and hamsters. To date, studies with the isolates from kuru and CJD brains have shown no fundamental differences from the strains of scrapie, except in restrictions in host range. More recently, rodent-adapted strains of kuru and CJD have become available. The only reliable assay system for these agents is bioassay, in which end-point titration is carried out. These agents are very hard to study because of the long incubation periods, with up to and more than 5 years in some systems.
1. Filterable to 25 to 50nm average pore diameter
2. Stable at 90oC for 30 minutes, but effectively inactivated by autoclaving. However, cases of CJD had been transmitted through the use of autoclaved instruments.
3. Hydrophobic with a strong tendency for aggregation of the infectious unit with itself and with cellular elements.
4. Continuum of size ranges from 40S->500S on rate-zonal sucrose gradients
5. Target size determined by ionizing radiation ranges from 64000-150,000 daltons
6. Gel filtration after zwitterionic detergent treatment gives <= 50,000 mol wt.
7. Density range 1.08-1.30 g/ml
The above physical properties suggest that the monomeric form of the virus is very small, but they must be interpreted cautiously. The known tendency of the virus for self-aggregation may yield falsely low values in irradiation inactivation studies.
1. Resistant or only partially inactivated by
aldehydes and related compounds (formaldehyde, glutaldehyde, B-propriolactone), Nucleases (RNAse, DNAse)
UV and ionizing radiation
Mild organic solvents (ethanol, xylenes)
Nonionic and nondenaturing anionic detergents
2. Moderately inactivated by
3. Effectively inactivated by
Autoclaving (134oC, 15 minutes)
Proteases (pronase, trypsin, proteinase K)
Denaturing detergents (SDS)
Strong chaotropic ions (thiocyanate, trichloroacetate)
Harsh organic solvents (phenol, chloroform, 2-chloroethanol)
Strong oxidizing agents (periodate, hypochlorite, permanganate)
Although they are highly resistant to many commonly used agents for disinfection, they are sensitive to a number of processes which are easily amenable for use in routine disinfection and sterilization.
1. Filterable and titrate cleanly
2. Replicate in high titres (up to 108 to 1010 infectious units per gram in brain)
3. Replicate first in lymph nodes, spleen, and thymus, and then in brain. Evidence for spread of virus along neural pathways, and from the spleen to the thoracic cord via autonomic nerves.
4. Restriction of host range and adaptability to new host
5. Strain differences (as judged by host specificity, incubation period, topography of brain lesions, ability to induce amyloid plaques, and the selection for different isolates from the same "wildstock" virus)
6. Genetic control of the host influencing susceptibility or resistance to infection.
7. Interference between slow-replicating and fast-replicating strains
8. Limited replication in tissue culture
9. Essential protein component for replication (as demonstrated by protease digestion; chemical modification by diethylpyrocarbonate, butanedione; inactivation by detergents such as SDS, ionic inactivation with guanidinium, thiocyanate, trichloroacetic acid; denaturation by urea and phenol)
These slow virus agents exhibited a number of conventional biological properties. They titrate cleanly from cell-free preparations, are filterable to 25 to 50 nm average pore diameter, and replicate in high titre in the brains of experimental animals. The kinetics of replication have been worked out in hamsters, from 107 LD50 to 1011 LD50. The earliest pathological changes are detectable 5 to 7 weeks after inoculation, and increase in severity during the clinical evolution of the disease. Routes other than intracerebral inoculation cause a remarkable increase in the incubation period, and it has been shown that the agent replicates first in the spleen and other tissues of the R-E system before establishing itself in the CNS. It is possible that the virus gains access to the CNS through a mechanism of neural spread along nerves in the spinal cord, then into the brainstem, cerebellum, and finally, the cerebral hemispheres.
On the other hand, the agents show a marked restriction of host range and the ability to adapt to a new host once the primary passage is achieved. Scrapie will readily passage in the same species but will only rarely "take" in other species such as mice, hamsters, and certain primates such as squirrel monkeys. All attempts to transmit scrapie to guinea pigs, rats, and certain other primates (especially the chimpanzee) have failed. Most primary cases of kuru and CJD will readily passage into nonhuman primates, including chimpanzees and squirrel monkey. Only in rare instances can kuru and CJD be transmitted to lower species such as mice, rats, hamsters, and cats. Upon successful primary passage, all viruses show adaptability in that the incubation period during the second and third passages, and then reaches a plateau. The incubation period can often be related to the dose of the inoculum. Attempts to culture these agents in tissue culture have met with limited success. Low titres of scrapie have been maintained in a mouse L cell line.
Different strains of the agent are thought to exist and their
replication and pathogenesis are controlled by a complex
interplay with host genetics. Certain breeds of sheep are quite
resistant to most strains of scrapie and different strains of
mice shoe significantly different incubation periods for the same
strain of scrapie and also show differences in the topology and
the nature of pathological lesions in the brain. Certain alleles
in sheep and mice are known to control the incubation period and
the susceptibility to infection and it is possible that these
alleles reside in the PrP gene.
1. Long incubation period
2. Lack of immunogenicity or host response
3. Chronic progressive illness without remission or relapse
4. Induction of spongiform change, neuronal loss, and gliosis
5. Absence of specific viral particle by EM
6. Specific nucleic acid not identified, and resistance to procedures that inactivate nucleic acids, such as low pH, nucleases, UV irradiation at 237nm, zinc hydrolysis, inactivation by psoralens and hydroxylamine
7. Unaffected by immunosuppression, immunopotentiation or antiviral drugs. Splenectomy may alter the course of infection
8. No CPE in tissue culture
9. Unusual spectrum of resistance to certain physical and chemical treatments
10. Induction of amyloid/scrapie associated fibril and conversion of normal host-encoded protease-sensitive protein (PrP) to protease-resistant homologue
The long incubation periods of these agents sets them apart from most other viral infections. The agents appear to replicate continuously from inoculation and manifest clinically when they gain access to the CNS and reach a critical titre. The complete absence of a detectable immune response is puzzling but this may be explained by the fact that the agent may be a modified host protein. Present evidence suggests that modified PrP protein is an integral, if not the sole component of the agent. It is uncertain whether there is any nucleic acid component. Certain physical properties suggest that a NA is not an integral part of the infectious particle, such as resistance to proteases, UV irradiation at 237nm, zinc hydrolysis, photochemical inactivation by psoralens and chemical modification by hydroxylamine. Some conventional viruses share one or more of these properties though. It is clear that any NA genome present would be extremely small (less than 30 bases), and would not code for the PrP protein.
Because scrapie infectivity is very resistant to heat and nucleolytic agents but sensitive to procedures that modify or destroy proteins, it is unlikely to be caused by a conventional virus. The unusual resistance of the infective agent to UV and ionizing radiation led not only to the conclusion that the mass of the agent is as low as 55K but also that it is devoid of nucleic acid altogether. Scrapie infectivity was purified several thousandfold from infected brain extracts by a procedure involving proteolysis under conditions where much of the protein but not infectivity is destroyed. The resulting preparations contain a protein of 27- 30K, designated PrP 27-30. The preparations are not free of nucleic acids but no scrapie-specific polypeptide has yet been identified, particularly no PrP sequence. PrP turned out to be host-specific protein, encoded by a single exon of a unique host gene. PrP is the product of a highly conserved gene found in organisms as diverse as fruit fly and man. It is a membrane bound protein thought to have an important, but yet unknown function.
Brains of scrapie-infected hamsters contain 2 forms of PrP; the cellular PrP and the scrapie PrPSc isoforms. Both proteins have a mass of 33-35K but they have different physical properties. PrPC is anchored to the cell surface and can be solubilized with ionic detergents as well as being susceptible to proteolytic agents. In contrast, PrPSc cannot be solubilized by ionic detergents and looses only an amino-terminal peptide to yield a protein of mass 27-30K called PrP 27-30. This is why PrP 27-30 is recovered from scrapie-infected but not normal brains. Monomeric PrP prepared from scrapie-infected brain retained infectivity. So far, no differences in the primary structure of PrPC and PrPSc have been detected, nor have any differences been found between PrP genes or mRNAs from normal and infected brains with respect to structure or copy number. The physical differences between the two proteins are therefore attributed to post-translational modification. It is known that PrP expressed in uninfected cells from the cloned PrP gene does not cause scrapie.
Strains of scrapie differ with respect to incubation time and brain vacuolation pattern, and can be passaged many times in the same inbred mouse strain and retain their characteristic properties. However, interconversion of certain scrapie occur reproducibly, particularly on transfer from one host to another. Incubation time is however, also determined by a host gene known as sinc or prn-i. It is thought that the prn-i gene may be identical with the PrP gene. Mice with short and long incubation times have different amino acids in positions 108 and 189 of the PrP protein, suggesting that the structure of the host PrP affects the incubation time. There are 2 main hypotheses for the nature of the prion;-
1. "Protein only" hypothesis - Prions are entirely
composed of protein material that can fold in multiple, structurally abstract
ways, at least one of which is transmissible to other prion proteins, leading
to disease in a manner that is epidemiologically comparable to the spread of
viral infection. The primary PrPC translation product would have to be converted
into the modified, infectious form in the PrPSc-infected cell. This conversion
may be catalyzed directly by PrPSc (Autocatalysis) Each scrapie strain would be
represented by a different variant of PrPSc which modifies one precursor into a
product resembling itself. Strain conversion on passaging in a different host
could be accounted for by polymorphic variants of the PrPSc gene.
In a more complex model, conversion of PrPC to PrPSc would be catalyzed by a host-encoded "converting enzyme" induced by PrPSc. The variety of scrapie species would be explained by a battery of distinct converting enzymes, each activated by the cognate strain of PrPSc.
2. "Nucleoprotein or Virino" hypothesis - the prion consists of a small nucleic acid and host-encoded protein. Strain differences are ascribed to variations in the nucleic acid, just as in the case of viriod RNA.
The overwhelming weight of available evidence at the present supports the "protein only" hypothesis. In 2 apparently unrelated families, one in the US and the other in the UK, Prusiner et al. found that an ataxic form of the Gerstmann-Straussler syndrome is linked to a change in codon 102 in one of the PrP alleles from proline to leucine. This mutation was not found in normal individuals or 15 individuals suffering from other forms of sporadic or inherited prion diseases. It is also thought that this change may play an essential in the pathogenesis of the disease. It is possible that in sporadic cases of GSS and CJD a somatic mutation gives rise to a variant PrP with enhanced capacity to convert into PrPGSS and PrPCJD. It is possible that in these individuals, the pathogenic PrPSc from arise spontaneously, although rarely. The other possibility is that they greatly enhance susceptibility for a "true" infecting agent. Additional mutations of the PrP gene have been found in other families prone to CJD or GSS.
When scrapie prions, which have been passaged continuously in hamsters are inoculated into mice, there is an incubation period of 500 days or more which diminishes to 140 days on the next passage in mice and then remain constant. A similar sequence of events take place in hamsters. The "nucleoprotein theory" attributes this adaptation to classical mutation and selection, whereas the protein only theory believes that the conversion of host PrPC by exogenous, heterogeneous PrPSc is a rare event, but once it occurs, the newly formed PrPSc, now being of the host type, catalyses further conversion both efficiently and rapidly. Prusiner et al. generated transgenic mice expressing the hamster PrP gene and inoculated them with hamster derived prions. The results were dramatic, incubation times were reduced from more than 500 days(in control mice), to 75 days in the transgenic mice. At the same time, susceptibility to the mouse-derived prions decreased. The agent produced in transgenic mice infected with hamster derived prions was highly infectious for hamsters but not for mice. Conversely, transgenic mice infected with mouse-derived prions yielded prions that were infectious for the mouse but not infectious for the hamsters. These results signify that hamster-derived prions readily convert the "transgenic" hamster PrPC but not endogenous murine PrPC into PrPSc. The species barrier would thus be due to the inefficiency with which PrPC is converted by the heterologus PrPSc.
A murine gene carrying the GSS proline to leucine change was inserted into mice. A founder male as well as 34 transgenic offspring expressing the mutant transgene developed neurological symptoms at 7 to 39 weeks of age and died within a month. The brains exhibited intense spongiform degeneration but, unexpected, despite the striking symptoms, PrPSc was not detectable. Mice overexpressing hamster PrPC or a normal variant of murine PrPC never developed symptoms. Brain extracts prepared from these transgenic mice transmitted CNS degeneration to some inoculated recipients. By inference, these results contends that PrP mutations cause GSS and familial CJD.
Thus the weight of evidence supports the protein only hypothesis. The main reason why the hypothesis is still met with reservation is the existence of prion strains. There are at least 2 distinct strains of murine prions (distinguishable by their incubation times), each of which can be propagated indefinitely on one and the same inbred mouse strain although there exists but 1 single PrP gene and the mouse is homozygous for this gene. Even more strains of scrapie have been reported. Therefore, PrPC would have to be converted into as many distinct conformational states as there are different prion strains. In the light of the new results, it is safe to conclude that PrP protein plays an essential role in the pathogenesis of spongiform encephalopathies and a modified PrP protein is at least part or whole of the infectious agent.
A study in 2015 concluded that multiple system atrophy (MSA), a rare human neurodegenerative disease, is caused by a misfolded version of a protein called alpha-synuclein, and is therefore also classifiable as a prion disease.