Download A virus behind the mask of prions?

REVIEW ARTICLE
Folia Neuropathol.
Suppl. B/2004 pp. 10–23
Copyright © 2004 Via Medica
ISSN 1641–4640
A virus behind the mask of prions?
Laura Manuelidis
Yale Medical School, New Haven, CT 06510 USA
The prion notion, that a host-encoded protein reacts with itself to become an infectious entity, has become
highly favoured as the causal agent in transmissible encephalopathies (TSEs) such as endemic scrapie of
sheep, epidemic bovine encephalopathy (BSE), and rare forms of human Creutzfeldt-Jakob Disease (CJD).
Much of the claimed “wealth of data” supporting the notion of prions, however, has been irreproducible as
well as contradicted by many experimental observations. By 1989, for example, it was already shown that
the “infectious prion conformation” could be eliminated without altering either infectivity or agent strain
characteristics. Efforts to make the prion protein infectious also continue to fail. All the biological features
of TSEs fit better with a conventional viral particle. Furthermore, structural data point to a spherical infectious particle of ~25 nm that contains a genome of 1–4 kb in length and probably encodes its own protective nucleocapsid. These viral features can account for the observed host recognition of TSE agents as
foreign. Although recognised, TSE agents, like many conventional viruses, can hide in a latent state for
many years in lymphoreticular tissues. Previous predictions made by this parsimonious viral interpretation
have been accurate, whereas the prion hypothesis has been confounded by several inexplicable realities
that have emerged. Recently developed TSE tissue culture models offer a way of identifying rapidly the
intrinsic and strain-specific agent molecules so needed for diagnosis and prevention.
key words: CJD, scrapie, BSE, transmissible encephalopathies, viral particles, commensal infections,
interference
“For this is so. Because”
Gertrude Stein, If I told him
INTRODUCTION
The epidemic of bovine spongiform encephalopathy
(BSE) in the UK, as well as the increasing spread of
a comparable infectious encephalopathy among domestic and wild cervids in the USA (chronic wasting disease
or CWD), make it important to resolve the nature of the
infectious agents that cause these neurodegenerative
diseases. Knowledge of intrinsic agent molecules can
facilitate rapid and sensitive diagnosis and ensure adequate preventive measures for both animals and humans. The infectious agents that cause transmissible
Address for correspondence: Dr. Laura Manuelidis
Yale Medical School, New Haven, CT 06510 USA
tel: 203–785–4442,
e-mail: [email protected]
10
encephalopathies (TSEs) typically lead to neurodegeneration only after a long asymptomatic period, with the
concomitant risk of transmission from apparently
healthy individuals [40]. This includes inadvertent person-to-person transmissions from tissue transplants,
blood [35, 36, 69] and possibly even by dental procedures [38]. The first positive blood transmissions from
animals and humans more than 25 years ago already
indicated that a larger population might silently carry
these agents than those expressing neurodegenerative
disease [35]. This is finally beginning to be appreciated
with the apparent second case of BSE-linked vCJD transmission by transfusion in 2004 [55a]. The observation
that infected blood carries the infectious agent to the
intestinal tract, in the opposite direction to that commonly assumed, also raises the likelihood of infected
Laura Manuelidis, A virus behind the mask of prions?
cells being shed in faeces even during the asymptomatic phase of infection [58].
Currently, the most favoured hypothesis in this field
is that the transmissible (infectious) agent is composed
of a host protein, known as the prion protein (PrP), which
becomes infectious by interacting with itself. This presumably infectious protein or “prion” form is defined by
its abnormal aggregation and by its resistance to limited proteolytic digestion in a test tube assay with detergents. This form is commonly designated PrPSc or PrP-res (equivalent terms), or PrP-amyloid. The other less
publicised hypothesis, based on many observations, is
that the infectious agent is a virus with its own independent genome. Like other viruses, all TSE agents depend
on host cells for entry, replication and maturation. The
purpose of this review is to reconsider briefly whether
the claims made for prions are justified, particularly in
the light of more recent evidence, and to consider whether the central data instead point to a more conventional viral particle that is unlikely to arise from the host
genome. This paper also specifies details of the most
probable TSE viral structure based on the evidence to
date and points out some old tricks that these viruses
probably perform. These predictions can be evaluated
experimentally and can thus facilitate the identification
of true causal candidates.
Conventional experiments that defined
the infectious particle. Are they still true?
TSEs such as BSE, Creutzfeldt-Jakob disease (CJD)
and scrapie are all caused by a class of infectious agents
that behave like viral particles, physically as well as biologically. These infectious particles have been reasonably characterised for their size and density but have
never been purified to molecular homogeneity. Hence,
as with any cellular viral preparation, host contaminants,
especially those from diseased brains, may never be
completely removed from concentrated or “purified”
infectious particles. Nevertheless, infectious TSE particles can be separated from many small cellular proteins,
including the majority of host-encoded prion protein
(PrP). Between 1989 and 1995 repeated sucrose gradient studies of sarkosyl-treated hamster CJD brain preparations revealed a discrete 120S peak of infectivity. In
contrast, > 75% of the abnormal PrP was concentrated
in non-infectious fractions of < 10S [65, 66]. Lectin chromatography studies had similarly shown a discrepancy
between PrP-res and infectivity; PrP was recovered
equally in two fractions, but one of these fractions contained ~100 fold more infectivity than the other [48]. Both
sedimentation and lectin chromatography data, therefore,
demonstrated that pathological PrP is not directly pro-
portional to infectivity. Thus they contradicted one of the
most essential arguments put forth to support the notion that abnormal PrP is infectious (Table 1). They also
show that PrPSc can be separated from infectivity despite frequent statements to the contrary. Subsequent
experiments with hamster brains infected by the 263K
(Sc237) scrapie agent in other laboratories reproduced
the same sucrose gradient separations of infectivity from
PrP-res [e.g., 61 and reviewed in 42].
Many additional animal and cellular studies further
confirm a lack of proportionality between PrPSc (PrP-res)
and the infectious titer. For example, Amphotericin B
treatment arrests abnormal PrP accumulation in hamsters infected with the 263K strain of scrapie agent but
does not prevent this agent from continuing to replicate
exponentially to very high titers [72]. The reverse has
also been reported. In salivary glands infectivity declines, while PrP-res accumulates [59]. Major discrepancies are also obvious in studies of two distinct human CJD strains transmitted to mice. Brain homogenates with 10-fold differences in PrP-res contain
10,000-fold differences in agent titers [43]. Notably,
these two very different CJD strains (like many other
distinct scrapie strains) show no distinguishing PrP-res
band patterns in the brain. Purified living microglia from
CJD-infected brain also have no detectable prions, yet
these myeloid cells contain maximal brain-equivalent
levels of the infectious agent [9]. Finally, GT tissue culture cells infected with a CJD agent show a progressive
650-fold increase in infectious titer, while PrP-res levels
remain constant [4]. The parsimonious interpretation
would be that the infectious agent is different from PrP.
To explain such discrepancies, however, speculative and
untestable differences in PrP folding as an intermediary and invisible PrP* form or some mysterious tertiary
conformation of PrP-amyloid that cannot be physically
resolved have been invoked [2, 56]. Thus an additional
revolutionary principle was conceived: some abnormal
PrP molecules are more equal than others.
Since no observable form of PrP can be used to predict infectious titers and there are no known markers
that are specific for the infectious particle, the purification of an agent is difficult and results are subject to a
difference in interpretation or emphasis. This is particularly problematic because in all infectious subcellular
preparations where reasonable amounts of starting TSE
infectivity is recovered, (i.e. > 10%) there are many other proteins as well as large amounts of chromosomal
DNA. A western blot that tests only PrP antibodies will,
of course, not reveal these other proteins [reviewed in 42].
Nucleic acids have also been dismissed as intrinsic
agent components (Table 1). Even in a recent paper that
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Folia Neuropathol., Supplement B/2004
Table 1. Major arguments and the “wealth of data” cited for “the fundamental principles of prion biology” [56]. PrP-res, aggregated PrP and PrP amyloid are the descriptive terms used for abnormal PrP, whereas PrPSc implies that these host-encoded
PrP molecules are the “infectious scrapie forms” or “prions”. Pathological PrP, also known as PrP-res because it is produced
by limited proteolytic digestion of cellular samples in detergents and leads to the formation of amyloid fibrils within minutes,
although this amyloid does not increase the infectious titer of the sample. “If it was so, it might be; and if it were so, it would
be; but as it isn’t, it ain’t. That’s logic.” (Lewis Carroll)
Claim
Experimentally
1) „PrPSc (Pr P-res*) is proportional to titer”
Comment
FALSE
Diverse data from many labs
TRUE
These also inactivate viruses
3) „No evidence exists for a virus-like particle”
FALSE
Discrete 25 nm viral particle
4) „Transmissible particles are devoid of nucleic acid”
FALSE
All infectious preps long nucleic acids
FALSE
Toxic pathology, but not PrPSc
2) „Procedures that modify or hydrolyze
PrPSc
5) „PrP gene mutations cause formation of
inactivate prions”
PrPSc”
6) „PrP gene mutations cause transmissible disease”
7) „Prion diversity is enciphered by
PrPSc”
Not reproducible
conformation
FALSE
8) TSE agent „strains can be generated by passing
through hosts with different PrP genes”
Sometimes
9) „No sign of an immunoresponse to foreign agent”
10) “Accumulation of
PrPSc
associated with pathology”
11) Protein X postulated to bind PrP for transmission
TRUE
PrPSc is late response to infection
TRUE
13) CJD infectious agent arises spontaneously
TRUE in textbooks
12
Often keep unique identity in different species
(as BSE)
Early anti-viral responses
12) “Prions defy the rules of protein structure”
If you don’t look for molecules other than PrP
you won’t find them
Several capsid-like proteins, in addition to a specific
endogenous retroviral capsid protein, can be detected
in infectious gradient fractions, along with nucleic acid
sequences up to 5kb in length. Co-sedimenting retroviral particles can be retrieved but these do not contain
TSE-specific information as determined by sequencing
[3]. However, their preservation and resistance to “treatments that hydrolyse or destroy nucleic acids” shows
that such treatments do not prove the existence of
Changing PrP-res folding has no effect
on strain or titers
FALSE
X not found yet
describes an infectious preparation as “purified”, silver stained nucleic acids are readily observed in infectious gradient fractions analysed on gels [63]. Micrococcal nuclease digestion can markedly reduce these
nucleic acids but it does not change the 120S sedimentation or the ~25 nm size of the infectious particles, nor does it alter their 1.28 gm/cc virus-like density
[64, 65]. Thus neither the virus-like particle size nor the
virus-like density of TSE agents can be explained by nonspecific binding of extrinsic nucleic acids. In fact, the
infectious particle behaves like a typical viral core, with
a nucleic acid genome covered by the armour of a viral
capsid.
Contamination
X is probably a virus
Possibly also of thermodynamics
An idea predating evolution
a prion without nucleic acids > 50 bases in length [56].
Moreover, the resistance of infectious TSE particles to
such treatments should not be surprising because viruses must negotiate some of the most hostile environments to reach their targets, for example through the
acids and enzymes of the digestive tract. Indeed, extensive nuclease digestions are frequently used to purify intact viral particles, such as those of hepatitis B and
poliovirus before extracting their small genomes. It is
also often argued that sensitivity to proteolysis is convincing evidence that the infectious agent is a prion.
However, protective viral capsids can be destroyed by
proteolytic treatments with consequent loss of intact
particle titers. Thus the nuclease resistance of infectious TSE particles, as well as their susceptibility to proteolysis, does not demand an “unprecedented” or new
biological principle of infectivity.
Agent nucleic acids in TSEs
The repeated presence of nucleic acids in all infectious preparations has already been summarised [42]
but, to bring this up to date, a recent experiment by Prusiner and associates should be noted [58a]. These experiments again repeat uncontrolled recovery and problematic detection methods but now admit 10–20 ng of nu-
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Laura Manuelidis, A virus behind the mask of prions?
cleic acid can be extracted from 263K infected scrapie
brain preparations with a total of 108–109 infectious
units (LD50). For a genome ~1,000 nt in length (the
length of hepatitis d), 1ng is sufficient to code for ~109
infectious particles. Hence tortuous assumptions and
calculations are used to exclude a viral genome. No attempts were made, however, to examine the sequences retrieved or to evaluate retroviral particle sequences
already shown to co-sediment non-specifically with the
TSE agent in more purified hamster 263K infected brain
preparations [42]. This requires only a simple and rapid
RT-PCR test with the primers already described [3]. The
preparative recovery of infectivity also seems to be quite
poor (~0.1% of the starting brain infectivity) because
“500 ml” of a 10% 263K infected brain homogenate
should have a total > 1012 infectious particles. Rather
than directly examining the nature of the nucleic acids
recovered, this paper instead re-explores less conclusive radiation effects, even though by now it is well established how several types of conventional viral particles are highly resistant to radiation [reviewed in 40].
This makes their conclusions about a lack of TSE nucleic acids suspect. In any case, the recovery of 10–20 ng
of nucleic acid from these new infectious scrapie preparations [58a] surely contradicts the statement that “no
detectable nucleic acids of any kind have been associated with prions” [57].
How compelling are the prion claims?
“A wealth of data” is often referenced to support the
contention that a host protein transforms itself into an
infectious agent [56]. A Lasker Award was first given to
Stanley Prusiner ‘for demonstrating how a genetic mutation can “misfold” ordinary proteins, turning them into
infectious agents that mimic viruses’, and such prionaffirming statements have become even bolder since
the 1997 Nobel Prize award held “no doubt” about the
existence of prions, “a new principle of infection”. Indeed, textbook chapters, largely written by Prusiner and
his collaborators, as well as recent papers from other
associated investigators, uniformly repeat a continuing
belief in prions that has become indisputable by popular stampede. It would also be difficult for a novice or
outsider to understand papers or reviews that are filled
with a new prion language that is misleading [42], sometimes in a seemingly purposeful way. For example, there
is the continued claim that prions have been injected
when instead a complex mixture of molecules, such as
a brain homogenate, has been used. Many papers also
start with the assumption that prions exist, so that the
experimental data and arguments are intentionally confined, in a circular fashion, to that assumption. It would
have been more impartial had these authors included
correctly cited data and the questions of those less taken by prions. A few recent definitive comments in the
abstracts or introductory paragraphs by “prion experts”
from this last year are quoted below, although it was
already apparent by 1992 that mention of any structural data that defined a conventional viral particle was
heresy [65]:
1) “The pathogenic PrPSc has the unique property of
being a self-replicating and infectious agent that
lacks nucleic acid” [54].
2) “There is considerable evidence that PrPSc is an infectious protein and that conversion of PrPC into PrPSc
is the central event in the propagation of prions, the
infectious agents in these diseases” [68].
3) “There is little doubt that the main component of
the transmissible agent of spongiform encephalopathies — the prion — is a conformational variant of
the ubiquitous host protein PrP(C) [71].
4) “The causative agent of transmissible spongiform
encephalopathies such as scrapie is PrPSc, a misfolded, protease-resistant version of the normal PrPC
protein [1].
5) “Prions are infectious pathogens principally composed of abnormal forms of a protein encoded in
the host genome. Remarkably, distinct strains of
prions occur despite absence of an agent-specific
genome: misfolded proteins themselves may encode
strain diversity” [17].
6) “Even now, despite the overwhelming evidence supporting it, some maintain that the infectious agent
must be a virus or a virino (agent containing its own
nucleic acid enveloped in host-encoded protein) or
that PrPSc must contain a small amount of host-derived nucleic acid (the “co-prion,” or molecule that
specifies prion infectivity). These alternative theories
are maintained even though, as with the miasma, no
one has ever demonstrated the presence of these
agents. It is demanded that the prion hypothesis satisfy the prion version of Koch’s postulate” [75].
Interestingly, not a single one of these commentators has, from their published reports, tried to purify the
infectious agent and most of them have little or absolutely no experience in transmitting different agent
strains.
Table I also shows additional prion-supporting statements [56] that have not been retracted despite good
evidence to the contrary. Most of these “compelling”
claims are self-explanatory but a few can benefit from
some expansion. Not only does PrPSc fail to predict infectious titers in brain fractionations, but other animal
and tissue culture experiments also show major discrep-
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Folia Neuropathol., Supplement B/2004
ancies. Even recent test tube amplifications that generate > 106 additional PrPSc molecules produce only
a minute amount of final infectivity, equivalent to 1–10
LD50 [16]. This amount is 10,000–100,000th of the infectivity in the original material with the same amount
and identical type of PrPSc. These “amplifications”, really PrP-amyloid conversions, are achieved by sonicating
infected brain spiked with normal brain in sequential
serial dilutions. In promoting this experiment as a “prion proof” one commentator even ignored his own published data showing how easy it is to carry a significant
number of infectious particles (105) on probes that are
analogous to those used for the sonication-dilutions [74].
PrP-res amplifications may become quite useful diagnostically but the ability of PrP-res to reproduce significant amounts of infectivity, and not just convert normal
PrP into PrP-amyloid, must be made more substantial
to constitute a prion proof. Other investigators have also
been unable to find significant replication of infectious
particles using the same published methodology [13].
It is also often stated that “unusual properties” of
PrPSc “mimic” those of prions. However, properties of
agent and PrP-res often diverge significantly. For example, the properties of PrPSc responsible for proteinase K
resistance do not correlate with those conferring thermostability on TSE agents [67]. Additionally, the heat
sensitivities for most of these TSE strains are quite conventional, with substantial inactivation of several strains
between 70 and 84°C. Many extraordinary inactivation
claims for TSE agents also centre on final sterilisation
levels of infectivity (to 0%) rather than on the inactivation characteristics of the more representative 99.9%
of infectious particles in the population. Finally, filtration data claiming a small prion size of < 100 kD and
< 15 nm were subsequently re-evaluated and found to
be due to detergent artefacts and leaky filters respectively [65]. Such irreproducible or flawed results, not
formally retracted by Prusiner, continue to be taken as
persuasively authoritative.
An infectious amyloid of one design
I would suggest that the evidence supporting prions is weak and oftentimes imaginary. The cumulative data continues to indicate that PrP abnormalities
are part of a secondary and pathological host response
to a foreign infectious agent. PrP-res is similar to other
reactive and often insoluble amyloids with a b-sheet
conformation. These fibrils accumulate in chronic diseases of heterogeneous cause and amyloid production and deposition appear to be part of a fundamental cellular response to perpetual stress. The prion
hypothesis therefore dresses PrP-amyloid with an in-
14
fectivity that is unique and then shifts the discussion
to causally unrelated neurodegenerative diseases such
as common forms of Alzheimer’s disease, designating
CJD and other TSEs as “pseudoinfections”, a rather
misleading prefix.
Although models for the conversion of membrane
proteins into an amyloid conformation may be applicable to disease progression and pathology in TSEs as
well as for other amyloid diseases, mechanisms of amyloid seeding or conversion seem largely irrelevant for
the essential strain-encoding molecules of the infectious
particle. One may wonder why, then, so few scientists
have attempted to characterise the infectious agent
rather than to follow all the twists and turns of the pathological PrP protein. Part of this may be due to the expense and length of animal bioassays for infection. This
impediment should be minimised with the new cell culture models for infection and the rapid tissue culture
assays that can distinguish specific agent strains
[e.g., 53]. However, there are other factors that have
also dissuaded, and possibly prevented, young investigators from wandering outside the prion box. These are
the major claims for prions that continue to be disproportionately emphasised, with great assurance, even
though some of these statements are clearly contradicted by data that has been reproduced, even in prion-centric laboratories.
Agents assert their individuality
in a PrP-res independent manner
The existence of distinct agent strains was, of course,
denied by Prusiner for many years, since the prion hypothesis could not easily explain the variety of isolated
agent strains. Even as late as 1997 he minimised the
number of TSE strains and states: “the primary structure of PrP-encoding prions during the passage history,
rather than the original source of inoculum, determines
strain characteristics in any particular host” [60]. The
epidemic BSE agent surely does not follow this rule. It
maintains its singular identity in every species it has
infected, even though the PrP pathology in those species is quite variable. Representative tested BSE linked
isolates from the numerous species infected, which include primates, felines, canines, gazella, kudu, caprines
and bovines, and thus far all yield the same BSE strainspecific profile in inbred indicator mice [14]. Moreover,
it has long been known that natural sheep scrapie
strains preserve their identity during serial passages in
mice, and can reinfect sheep to produce the original
scrapie incubation and neuropathology characteristics
regardless of the PrP differences between these two
species [73]. The vast majority of distinct scrapie strains
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Laura Manuelidis, A virus behind the mask of prions?
also provoke no PrP-res differences when propagated
in inbred mice and in hamsters there is only a single
TSE strain, isolated from a mink, that is selectable and
provokes a different brain PrP-res pattern than the various other scrapie agents [11, 60]. Finally, PrP-res banding patterns can be different in brain and lymphoreticular tissues of a single animal, but both of these tissues
transmit only one and the same agent strain. Nevertheless, one is instructed that PrP must “encipher” and propagate individual strain properties (Table I), while experimental data to the contrary are assiduously ignored.
In an experimental setting PrP folding and glycosylation can be demonstrably abolished. These experimental manipulations show that PrP-res folding patterns and
glycosylation features are irrelevant for determining
strain properties, since these modifications have no
effect on either the infectious titer or on the propagation of strain specific properties. Such experimental
manipulations include, for example, the unfolding abnormal PrP that can be produced by small changes in
buffer and dispersion conditions, so that PrP-res characteristics are no longer present and the abnormal PrP
is now rapidly degraded by proteinase K, unlike the untreated control material. In fact, the only change accompanying this PrP unfolding is a slight elevation in titer,
one that is consistent with disaggregation of infectious
particles rather than with the prion predicted loss of
infectivity [66]. Enzymatic removal of sugars also has
no effect on infectivity or strain characteristics of a CJD
agent [48]. More recently PrP modifications produced
by chronic growth of different agent strains in cell cultures with unique PrP patterns also produced no change
in strain-specified properties. In these cultured cells, PrPres folding and glycosylation patterns are markedly different from those in infected brain, yet two very distinct
CJD agents maintained their original strain behaviour
in this setting, as shown by inoculation of these chronically infected cells back into mice [4]. In other words,
TSE agents breed true regardless of the cell specific
PrP glycosylation pattern or by the post-translational PrP-res folding patterns these agents provoke. In fact,
agents maintain their specific identities, whereas the
pathological PrP characteristics are determined by cell
type as well as by species.
Is any form of recombinant PrP infectious?
Correlations are ultimately unsatisfying for drawing
a reliable conclusion about the nature of these causal
infectious agents. In principle, the true TSE infectious
agent must be able to fulfil Koch’s postulates, although
in some cases, such as HIV and CJD infections of humans, it must draw on analogous experimental infections
in animals. In the case of a TSE virus this proof must be
made with a purified viral particle or preferably, if possible, with the naked nucleic acid genome. With a viral
genome there can be no question of residual prion protein. Similarly, the most convincing way to prove the prion hypothesis is to recreate bone fide transmissible disease with some form of recombinant PrP (recPrP). The
latter has been attempted repeatedly for > 20 years without success, and perhaps that was the impetus for postulating protein X, a co-factor needed for PrP infectivity or
for designating TSEs as “pseudoinfections”.
Only in the past year has there been a claim that
a truncated recPrP is capable of infecting mice [29].
There are several odd features in this report that make
this proof less than overwhelming. Firstly, two differently treated preparations of recPrP both formed amyloid
fibrils in a test tube, yet only one of these resulted in
some spongiform change (1° passage), a change often
seen in uninoculated transgenic (Tg) mice with more
than normal PrP copies. Secondly, these primary passage mice became ill only after > 380 days, a rather
weak effect and one that would not be expected in recipient mice carrying multiple copies of the identical
truncated PrP sequence as the recPrP fibrils. Thirdly, of
the many mice that were injected with recPrP, only one
primary passage Tg mouse brain homogenate produced
spongiform changes when injected into recPrP Tg mice
for 2° passage. Fourthly, these 2° passage mice, with a
shortened incubation time of ~250 days, suddenly
showed a very different distribution of brain lesions.
These lesions were remarkably similar to those produced
by the common RML scrapie agent used in that laboratory. According to prion theory, “artificial prions” should
produce “novel properties not found in nature” and this,
therefore, was not apparent. Furthermore, such a lesion
profile change is quite atypical in 2° passages, particularly for a new prion strain that should be enciphered by
this truncated PrP. Fifthly, on the 3° serial passage the
likely truth of RML contamination asserts itself. Both wt
and Tg mice show a characteristic short RML incubation time of < 150 days, again with an RML lesion profile. Even in cross-species TSE agent transmissions,
there is only a gradual and small reduction in incubation time after the 2° passage, as documented in rat
CJD transmissions [44], as well as in > 30 independent
transmissions of human CJD to guinea pigs, mice and
hamsters [33, 39, 47].
Thus these recPrP results are more consistent with
an RML contaminant, possibly made during removal or
preparation of the first mouse brain, than with the conclusion that a truncated recPrP produced a new infectious TSE strain that suddenly altered its PrP conforma-
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Folia Neuropathol., Supplement B/2004
tion. The claim that new strain characteristics were acquired is also logically inconsistent, since the RML agent
characteristics were apparent in both the wt and truncated PrP Tg mice. If PrP enciphers the agent strain,
each of these mouse genotypes should have produced
its own unique strain. Denying this possibility is as unhelpful as ignoring other contaminations in experiments
cited to prove there are PrP “genetic” forms of TSE (see
below). Several investigators, including those who believe in prions, have also expressed some of the same
reservations about contamination, as well as the inevitable toxic spongiform pathology in PrP multi-copy Tg
mice [e.g. 16, 18, 52]. Such reservations, however, did
not arrest the inflation of these results by some prion
proponents for “the birth of a prion” [70]. Along with
other prion claims, they seem more exemplary of Languimer’s description of pathological science based on
small effects [27]. It would have been more convincing
if the recPrP directly infected a wt mouse to produce a
distinct TSE agent strain in < 350 days.
PrP, a host susceptibility factor linked
to pathology
The evidence does, on the other hand, clearly show
that host PrP is essential for susceptibility to, and modulation of, infection (Table I). This requirement for host
PrP (but not PrP-res or PrPSc) was ultimately proven by
experiments in PrP knock-out mice where a scrapie infectious agent failed to propagate or survive [15]. Propagation of murine passaged CJD agents was also similarly negative in these mice (LM unpublished data). Similarly, administration of PrP antibodies, since they can
remove or mask host PrP, can also retard disease [32].
These data showing that host PrP is essential for TSE
agent propagation are comparable to the requirement
for particular cellular receptors by many viruses where
even single amino acid changes in a host protein sequence can prevent infection [reviewed in 40]. The PrP
knock-out paper only incidentally considers that the TSE
infectious agent might be “something other than PrPSc”,
or the possibility that host PrP might be an essential
viral “receptor”, as suggested many years before
[47, 48]. Maybe this old concept, like those of TSE agent
strains [55], completely asymptomatic infections [20],
the involvement of the lymphoreticular system by TSE
agents [22] and the horizontal transmission of a prevalent environmental pathogen to more genetically susceptible mammals may also, one day, be newly rediscovered, along with the intrinsic cleverness of viruses.
Old-fashioned viruses, much as TSE agents, can target
lymphoreticular cells, hide in a latent state for many
years and can also be transmitted vertically. Thus sev-
16
eral types of virus can easily account for some cases of
TSE familial disease, although only PrP germline mutations have been considered.
Classification of TSEs: familial
and “spontaneous” TSEs
The prion classification of TSEs has been etched
deeply in the TSE literature of the last 20 years. However, there is still no reproducible experimental evidence
that any PrP mutation can cause or create an infectious
agent in any animal. The argument that familial CJD is
only explicable by the prion hypothesis is flawed on several counts, aside from the fact there are viruses, such
as retroviruses, that integrate in the germline. Firstly,
the “largely circumstantial genetic evidence” for prions
[2] is limited to primary data on very few people in very
few CJD families. This family data is equally compatible
with an enhanced susceptibility to specific TSE agents
in the environment. Indeed, the common scrapie strain
in Europe is so widespread that sheep with more susceptible PrP genotypes carry a risk of > 20% infection
[12]. This number approaches the 50% incidence ideally found in purely genetic dominantly inherited noninfectious diseases.
Secondly, experiments to prove that genetic PrP mutations can cause infection have been fruitless. The claims
that the Gerstmann-Sträussler Sheinker (GSS) 102L PrP
mutation is sufficient to either induce PrPSc or to cause
transmissible disease are untrue. A negligible amount of
PrP-res was made by changing the protease digestion
conditions and similar bands can be produced in normal
brain homogenates under the same conditions (LM, unpublished observations). Furthermore, those 102L PrP
mice transmitted only the uniquely fast laboratory 263K
hamster agent, and this transmission was considered
a contaminant or “very weird” by other investigators
[in 44, 50]. These hamster transmissions remain curiously unmentioned in recent experiments to prove the
transmissibility of recombinant PrP [29]. Most importantly,
neither infection nor PrPSc forms could be reproduced in
transgenic mice with normal levels of the 102L GSS
mutation [10]. Even the “spontaneous” spongiform changes originally published [24] have been shown to be due
to toxicity from the freakishly high copy numbers of the
inserted 102L PrP transgene. Thirdly, a single and probably unique TSE agent should be determined by this 102L
mutation. Instead, geographically distinct CJD agents,
including the common sporadic CJD isolate, infect GSS
patients with this 102L mutation [53]. In sum, the evidence for a genetic PrP infectious element remains unfounded, although the exact nature of the familial inheritance pattern remains undetermined.
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Laura Manuelidis, A virus behind the mask of prions?
It is, furthermore, clear that natural scrapie in sheep
is neither a spontaneous nor an inherited infection.
Sheep with the more susceptible PrP genotype and
a high incidence of scrapie in the UK do not spontaneously develop scrapie when bred in Australia, a scrapiefree country [25]. Some observers have suggested that
scrapie agents may be transmitted by a vertical or by
a transplacental route in sheep. Vertical and transplacental infections have not been detectable in experimental CJD when offspring are conceived by CJD infected parents, as observed for up to 12 years in guinea
pigs [37]. The disappearance of human kuru is also incompatible with either maternal or vertical transmission
[23] and was not observed in normal mice or in primates
[34]. Recent observations on natural scrapie may resolve this discrepancy, at least in part. Investigators point
out that it is not always possible to discriminate between
maternal transmission and horizontal transmission by
close contact during the perinatal period but ongoing
studies indicate that the perinatal period is a dangerous time for susceptible lambs [28]. Although some
apparently vertical sheep scrapie transmissions might
be due to variations in ovine versus primate or guinea
pig immunological development [37], it remains possible that some members of the general class of TSE
agents may be highly prevalent, though rarely pathogenic, and may also be passed vertically or transplacentally in particular species. Thus, in assessing potential viral structures, one may find some agents in this
class that are typically commensal and non-pathogenic
and possibly able to be transmitted from a parent. This
is not unprecedented. There are a number of normally
non-pathogenic commensal viruses, such as the JC
papova virus, that persist in the host for a lifetime and
we have also suggested that some strains of CJD may
be prevalent, but non-pathogenic for many years [e.g.,
34, 39, 46]. Such a possibility appears more probable
now with the observed protection afforded by particular CJD agents against a variety of TSE agents [53].
One is then left with a failure to show that any genetic PrP has been able to cause a TSE. Moreover, the
postulate of a spontaneous PrP mutation or conversion
rather than the presence of an environmental agent
must be limited to sporadic CJD cases. Oddly, this assumption, that sporadic CJD is a “spontaneous neurodegenerative disease” creates a new category, unlike
any other naturally occurring TSE [57]. The spontaneous generation of an infectious agent is also entirely
speculative, logically inconsistent [42] and thermodynamically improbable. Moreover, the clustering of CJD
cases has been consistent with exposure to an environmental agent of low virulence [30] rather than a ran-
domly generated pattern. Spontaneous generation of
infectivity has also has been used, conveniently, to remove any responsibility for the spread of BSE in Europe
(“an act of God”) and for years of slow governmental
actions. The spread of this suddenly evolved and more
virulent BSE agent to humans, as well as to many other
species, was also unanticipated by Prusiner because it
was not consistent with his proposed mechanisms of
like-to-like PrP interactions for prion propagation. Such
BSE cross-species infections were, however, quite an
obvious possibility [41] from our many successful transmissions of CJD across species that bore highly divergent PrP sequences [34, 47]. Although like-to-like host
PrP sequences may be needed to convert PrP to PrP-res amyloid, this interaction and its consequences are
epiphenomena that are insufficient, and probably not
required, for the replication of infectious particles.
Immunological parameters of TSE infection
A classic experiment of Hadlow and his associates
in 1967 showed that a scrapie agent inoculated intramuscularly first replicated in lymphoreticular tissues
such as the spleen before passing to neural tissues such
as the spinal cord and cerebrum [22]. This is a typical
route of dissemination for the vast majority of known
human viruses, including those such as poliovirus that
eventually spread to the CNS and those that eventually
evade immune recognition. The presence of infectious
TSE agents in myeloid cells such as migratory macrophages, microglial cells and dendritic cells [6, 9, 49], therefore, recapitulates this viral pattern of tissue preference
and progressive spread. Moreover, blood and reticular
tissues provide a conduit and source for latent accumulation, as well as for subsequent reactivation and
agent dissemination as previously proposed [42]. The
original observations of Tateishi’s group also showed
that specialised follicular dendritic cells (FDC) of the
spleen were probably infected, since pathological PrP
was associated with these cells [51]. PrP-res largely
accumulates at the surface of the FDC during infection
[49], a place known to trap several types of viruses including HIV. Although the dependence of different TSE
strains on FDC can be variable, as determined by the
infection of Lymphotoxin-b and other relevant knock-out
mice [5, 26, 49, 62], FDC are involved in all the experimental TSE models examined thus far. FDC and antigen-presenting myeloid cells are built to recognise
a foreign agent and would be a likely stopping place for
a persistent or evasive virus. In contrast, this lymphoreticular route and cell-specific involvement by TSE agents
seems highly improbable for a host protein that is more
abundant in other peripheral cell types and tissues.
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17
Folia Neuropathol., Supplement B/2004
The presence of TSE agents in myeloid cells also
raised the issue of an immune system response to TSE
agents, despite frequent blanket dismissals of this possibility. Thus we began to reinvestigate adaptive immune, inflammatory and innate immune responses in
experimental CJD, realising that a lack of evidence in
this field generally means a lack of experiments. Gadjusek and his associates showed there were no neutralising antibodies in CJD and our own tests on agent-inhibitory serum factors in animal interference experiments
are in accord with this conclusion [46]. Nevertheless,
neutralisation is a rather insensitive test of adaptive
immune system responses and an additional few cursory serum tests against blotted brain proteins are insufficient to exclude the possibility that the immune
system could recognise and respond to a TSE agent.
Moreover, a lack of neutralising antibodies does not rule
out a virally encoded capsid or other antigenic protein,
because many latent and persistent viruses evade neutralisation and/or suppress cell-mediated antibody responses. HIV is an obvious example of a virus that provokes antibodies that are ineffective in protecting the
host. Without the isolation of more purified TSE-infectious particles and their molecular characterisation for
intrinsic agent antigens a host antibody response to
these agents may be overlooked. Thus, for the time
being, it seems best to remain open-minded about the
possibility that a virally encoded (non-host) TSE specific
antigen may, in some circumstances, elicit an antibody
response that can be used to follow TSE infectious particles. This possibility seems more prudent now that
there is substantial new evidence that the host recognises TSE agents as foreign.
The prion hypothesis rests on the claim that, since
the agent is a prion and since self-encoded PrP and
PrP-res provoke no antibodies, the TSE agent (as well
as its presumed, and as yet undetected, host-encoded “protein X” that “chaperones” infectivity) must also
be incapable of provoking any host immune response.
The early microglial recruitment in rat CJD that precedes PrP-res, however, first suggested that the host
might respond to TSE agents as they do to other foreign viruses that evade immune destruction [44]. Thus
we began to examine inflammatory responses in CJD-infected versus uninfected microglia and found many
virus-like inflammatory CJD changes that were not
mimicked by the application of large amounts of PrP-res. PrP-amyloid, in fact, produced almost no microglial response in contrast to the application of standard inflammatory stimulators [8]. By following a number of these CJD agent-induced microglial markers in
the whole brain, we also found that some of these in-
18
nate immune and inflammatory responses were activated at substantial levels (5 to > 40 ¥ normal). Relevant molecules, including those in the pathway of interferon production, were readily apparent by standard
RT-PCR tests [7]. There was also a pattern of response
to CJD infection that not only precedes PrP-res by 60
days, but can be specific for the stage of infection as
well as the specific agent strain [31]. The host recognises the invading agent as early as 20–30 days after
inoculation with inflammatory and innate immune system responses, while PrP-res first appears at 90 days.
These types of innate immune responses are often
seen in many covert viral infections and even at endstage disease are different from those in Alzheimer’s
disease. Thus the touted lack of a host response to
TSE agents (Table I) has been refuted by evidence, and
this opening can provide information that is useful for
both diagnostic and therapeutic approaches to infection and disease progression.
A useful example of host recognition:
interference in vivo, and the rapid diagnosis
of strains by culture
The paradigm of viral interference has also been
tested extensively in mice with two very different CJD
agent strains. Interference occurs when an infection by
one agent strain prevents superinfection by a second
related challenge agent. We first tested the ability of
a slow and avirulent strain of CJD, typical of sporadic
CJD isolates, to prevent superinfection by a more virulent Asiatic CJD isolate in mice [43, 46]. The results of
these experiments were surprisingly dramatic. With low
doses of the protective agent mice lived free of disease
for their 2 year normal lifespan, even after intracerebral challenge with the second agent. Unprotected mice
all died ~350 days earlier. Remarkably, PrP-res was not
involved in this protection since PrP-res remained undetectable during this prolonged time of challenge. Furthermore, brain factors but not serum factors appeared
to be involved in protection, a finding consistent with innate immune responses provoked by the first protective
agent. It was also possible to determine in these long
incubations that the simultaneous propagation of two CJD
strains in a single animal produced no “chimeric” or intermediary TSE strain as predicted by the prion hypothesis. Instead, doubly infected brains showed that each of
the two strains bred true with no mixed phenotype [45].
Prion proponents have not explained these positive interference results, shown in several experiments, and
using different routes of challenge (such as ic and iv),
although they appear to be inconsistent with some form
of a purely host-encoded infectious protein.
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Laura Manuelidis, A virus behind the mask of prions?
Figure 1. Basic strategy to test interference against secondary superinfection by a challenge TSE agent.
Interference in animals can be based on a multicellular and complex series of responses. To find if neural cell cultures that are free of immune system cells
could mount an interference effect we developed a rapid, more flexible, and simple co-culture test that could
be used to evaluate a variety of CJD and scrapie agent
strains that had similar incubation times in mice [53].
Figure 1 shows the basic co-culture strategy. These experiments demonstrated that 1) interference clearly
occurs in non-myeloid cells, 2) sheep-derived scrapie
strains can interfere with human derived CJD agents
and vice versa, and 3) interference is dependent on the
individual agent strain, but not on the presence or absence of pathological PrP. Notably, two scrapie strains
showed very different abilities to protect cells from superinfection by a CJD agent. Furthermore, the amount
and pattern of PrP-res was completely irrelevant for this
interference. PrP-res was also not necessary to prevent
superinfection, since cells infected by an agent that provokes no PrP res were resistant to challenge by both
CJD and scrapie agents. Agents that induced very large
amounts of PrP-res also failed to determine the protection observed, as would be expected for PrP competition. Indeed interference depended only on the continued presence of the infectious agent, as demonstrated
by “curing” experiments. Whatever the exact mechanisms of interference, these data point to innate host
responses that are specific for particular sets of TSE
agents and/or viral products that can restrict particular
challenge agents. Either or both of these mechanisms
may limit the entry or replication of challenge TSE particles or may enhance their clearance.
Animal interference assays take more than a year.
In contrast, these recently developed cell culture models can reveal strain specific characteristics within 25
days, and can be used to discriminate between strains
with similar incubation times in mice. Hence one can
begin to test whether a relatively non-pathogenic agent
of humans, such as the common sporadic CJD agent,
can be a factor in preventing widespread scrapie and
BSE agent infections in people. Indeed the overall interference data are entirely consistent with the presence
of one or more environmental TSE agents that are commensal but rarely pathogenic. Susceptible tissue culture models can, additionally, provide the essential
means for rapid screening of infection. Thus they are
likely to be useful for testing agent purification procedures, for assessing infection during asymptomatic
phases of disease when PrP-res is not detectable and,
possibly, for examining cells and fluids with low agent
titers, such as blood or CSF. The quantitative limits of
such cell culture agent titrations are currently being
evaluated. Such rapid quantitative assays are sorely
needed, especially because abnormal PrP, although
a good disease marker, is inadequate for following the
infectious agent.
Some infected cell lines display relatively high infectious titers, similar to those found in end-stage brains
[4]. Thus one should, additionally, be able to purify TSE
agents from cultured cells more effectively without the
complications of neurodegenerative changes in a complex tissue such as the brain. The rapid determination
of specific agent strains by co-culture is also invaluable
for assessing uncharacterised infectious isolates and
sudden changes in a strain in response to treatment,
as well as performing more mundane checks of inadvertent contaminations. Finally, the development of
TSE agent-susceptible cells provides a chance to evaluate effectively small amounts of pure molecules that
would be degraded shortly after inoculation into animals. These molecules may include specific naked
nucleic acid sequences as well as recombinant PrPs.
These cell-based infectivity assays are more biologically meaningful than test tube manipulations under
non-physiological conditions to produce PrP-res. Cells
that show the hallmarks of infection after the intro-
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19
Folia Neuropathol., Supplement B/2004
duction of suspect molecules can also be inoculated
back into animals, ultimately to prove which ones faithfully fulfil Koch’s postulate. In animals the infected cells
should produce both transmissible infection and
a specific disease phenotype, as do cells infected with
impure agent preparations [4]. This seems the most
objective way to determine intrinsic agent molecules,
viral, prion or otherwise.
Features of a TSE virus: predictions based on the
biological and physical/molecular data
Although no one would be foolish enough to rule out
any hypothesis in the TSE field at this point, it is time to
propose the most likely TSE agent structure from my
conservative point of view, based on the reproducible
observations to date. This proposal is meant to encourage experimental tests for validity and, hopefully, arrest
the facile and vague dismissal of anyone who dares
question the reality of a prion. Additionally, predicting
the features of a TSE virus can be helpful since it provides specific guidelines for judging if an isolate or structure obtained is a reasonable causal candidate. The
following biological features are based on properties
briefly reviewed above and indicate:
1) TSE agents will be in a class of viruses that contain
commensal and non-pathogenic members.
2) These viruses will be likely to infect, or be found in,
a variety of species, although some strains may be
largely restricted to a single species.
3) They will not be endogenous viruses but will be foreign and recognised, though not necessarily efficiently eliminated by host defence mechanisms.
4) They will probably code for at least one virus-specific (non-host) antigen.
5) Some members of this viral class may have the capacity to be transmitted vertically.
6) Non-specific stress, as well as other infections and
diseases or even ageing itself [39], may allow the
non-pathogenic commensal agents to recrudesce
from a latent carrier state to one causing disease.
7) The more virulent TSE-specific viruses should induce
abnormal PrP in susceptible cells and/or animals.
8) Some of these agents may cause progressive infection in a small percentage of the human population
without any quiescent non-pathogenic period.
Before continuing with the most likely physical features of TSE viruses, it is important to cite additional
refining experiments and calculations. In order to determine the physical size of the agent more precisely,
we analysed the nuclease-digested 120S infectious
peak by field-flow fractionation in 1992. Infectious particles co-migrated with spheres of 25–30 nm [65], and
20
thus it is likely that the CJD agent is a “round” 25 nm
particle. High-pressure liquid chromatography analysis of the 120S infectious peak additionally revealed
the infectious agent has a molecular weight of 106–
–107 Daltons, since it eluted just before the thyroglobulin marker of 0.6 ¥ 106 Daltons [65]. Electron microscopy further confirmed the presence of 25 nm particles. Figure 2 shows the 25 nm particles in thin sections of the 120S peak after dilution of the sucrose
and pelleting of the material at 100,000 g for 1 hr
(LM, unpublished data). An embedding method was
chosen in order to have a fair representation of the
material. In contrast, wet preparations can differentially
bind to grid surfaces. The virus-like particles shown were
not seen in parallel normal brain fractions and were not
labelled by PrP antibodies or by wheat-germ agglutinin
lectin when spread on grids, unlike preparations that
were enriched for abnormal PrP. If these 25 nm parti-
Figure 2. Peak infectious 120S fraction from a micrococcal
nuclease-treated hamster CJD concentrate treated with micrococcal nuclease before loading on sucrose gradient. Thin
section shows two adjacent 25 nm particles (filled arrow) as
well as possible particles cut on edge (as at open arrow). Pellet material fixed with 1% gluteraldehyde, osmified and embedded in Epon. Bar Is 100 nm.
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Laura Manuelidis, A virus behind the mask of prions?
Table 2. Likely physical, molecular, and pertinent biological features of TSE agents from experimental data
Predicted viral features
Comments
25 nm “round” (as dodecahedral) particle
By field-flow fractionation & morphological particles
Sedimentation of ~80S in sucrose
120S before removing extra PrP
& ~2 ¥
106
Daltons after PrP removed
By HPLC of 120S sucrose peak
Genome `1–4 kb in length
Possibly encoding capsid and/or viral polymerase
TSE-specific members
May bind PrP-rich membranes to induce amyloid
Preference for brain and lymphoid tissues
From group of related viruses with different tissue preferences?
Pathogenic and non-pathogenic members
Different strain virulence for various species, PrP genotypes
Commensal members of class
As latent, persistent or non-pathogenic infections
cles are the virus, as I suspect, then their failure to bind
PrP antibodies and their lack of PrP glycosy, residues
indicate that a protein other than PrP is part of their
protective shell. Indeed the virtually universal assumption that PrP must be an intrinsic part of the infectious
entity may not be true. A 25 nm particle size can accommodate a genome of ~1–4 kb, a length sufficient
to code for at least one protein such as a protective
nucleocapsid.
Additional treatment of the 120S peak by sonication in 0.5% SDS further demonstrated that almost all
of the residual PrP in this fraction could be solubilised,
whereas the 25 nm particles remained intact and sedimented at 100,00 g ¥ 1 hr with quantitative recovery
of infectivity. There were no methods to show directly
that the 25 nm particles contained nucleic acids. However, in contrast to SDS disaggregation of infectious
particles, more disruptive Gdn-HCl treatments that solubilised particle nucleic acids destroyed infectivity as
well as the sedimentation of the 25 particles [48a]. Interestingly, multimeric particles of PrP remained after
these disruptive treatments, even though infectivity was
destroyed. The prion hypothesis predicts that a PrP dimer
or tetramer should be sufficient for infection. These
purifications not only undermine the prion structure proposed, but also allow one to estimate the likely physical
parameters of a TSE virus. When one accounts for the
removal of the residual PrP aggregates by sonication in
SDS, as well as the ability to recover nucleic acids up to
5kb in length [3], it seems reasonable to predict that
this CJD agent will resolve as:
1) a “spherical” (as in dodecahedral) viral particle of
25 nm;
2) with a sedimentation of ~80S (range 60–120S);
3) a molecular weight of ~2 ¥ 106 Daltons, (range 106–107);
4) that contains its own nucleic acid genome of ~1–4kb
in length;
5) and codes for its own protective nucleocapsid and,
possibly, also a viral polymerase.
These physical features, as well as key biological
properties of TSE agents, are listed in Table 2. Needless to say, a genome of 1–4 kb in length is more than
sufficient to encode specific agent strains, in addition
to at least one functional protein.
Field-flow fractionation has now also been done on
RML scrapie-infected brain preparations, and infectivity had a comparable particle size of 17–27 nm [63].
The scrapie peak of infectivity was broader than in our
studies, possibly because those infectious particles were
not first separated from the bulk of pathological PrP.
Those scrapie fractions also had a large amount of higher molecular weight silver stained material on gels, consistent with nucleic acids that might broaden the size
distribution of field-flow fractions, but the loaded “purified” infectious preparations were apparently not analysed for nucleic acids. The authors’ interpretation, as
well as that journal’s editorial comment in that issue,
centred on the size of PrP required for infectivity [63].
CONCLUDING REMARKS
Although the preceding observations weigh in
favour of a viral structure made by a viral genome with
at least one virally encoded non-glycosylated functional
protein, it should be emphasised again that the above
predictions are proposed as a working hypothesis that
can be systematically tested. It should also be noted
that, although the overall data on TSEs make a prion
quite improbable, they are still insufficient to exclude
host PrP as the “essential protective coat” for a scrapiespecific “small informational molecule” as proposed
in the virino hypothesis [21]. The physical evidence to
date also does not address the idea of some host-encoded nucleic acid co-factor [19] or a “protein X” as
essential for infection, although the biological proper-
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21
Folia Neuropathol., Supplement B/2004
ties, such as host recognition of these agents as foreign and the environmental exposure that is required
for scrapie infection, are more simply explained by
assuming the presence of a foreign viral particle. Cell
culture models provide a new opportunity to perform
independent experiments to elucidate the intrinsic
molecular components of these agents. Susceptible
cell cultures can be used for simplified agent purifications, agent titrations and strain-specific assays. These
newly effective approaches to TSEs should open the
field to those who are curious and fearless enough to
explore what is.
ACKNOWLEDGEMENTS
I thank Sheldon Penman for his encouragement and
suggestions. This work was supported by NIH grant
NS12674 and DOD grant DAMD-17-03-1-0360
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