Sludge Watch ==> 25 nm Virion Is the Likely Cause of Transmissible Spongiform Encephalopathies

[Maureen Reilly]

Sludgewatch Admin:
We are hearing again and again that 'prions' may not be the infective agent 
for diseases like 'mad cow disease' BSE, and 'Chronic Wasting Disease' CWD, 
and Creutzfeldt Jacob Disease.  Instead, this research points to a slow 
acting virus or virus like agent.

PDF file is attached.

Or...if you fear attachments you can read it below.

.................................................



Journal of Cellular Biochemistry 100:897–915 (2007)
A 25 nm Virion Is the Likely Cause of Transmissible
Spongiform Encephalopathies
Laura Manuelidis*


Yale Medical School, New Haven, Connecticut 06510

Abstract The transmissible spongiform encephalopathies (TSEs) such as 
endemic sheep scrapie, sporadic human
Creutzfeldt-Jakob disease (CJD), and epidemic bovine spongiform 
encephalopathy (BSE) may all be caused by a unique
class of ‘‘slow’’ viruses. This concept remains the most 
parsimonious explanation of the evidence to date, and correctly
predicted the spread of the BSE agent to vastly divergent species. With the 
popularization of the prion (infectious protein)
hypothesis, substantial data pointing to a TSE virus have been largely 
ignored. Yet no form of prion protein (PrP) fulfills
Koch’s postulates for infection. Pathologic PrP is not proportional to, or 
necessary for infection, and recombinant and
‘‘amplified’’ prions have failed to produce significant infectivity. 
Moreover, the ‘‘wealth of data’’ claimed to support
the existence of infectious PrP are increasingly contradicted by 
experimental observations, and cumbersome speculative
notions, such as spontaneous PrP mutations and invisible strain-specific 
forms of ‘‘infectious PrP’’ are proposed to explain
the incompatible data. The ability of many ‘‘slow’’ viruses to 
survive harsh environmental conditions and enzymatic
assaults, their stealth invasion through protective host-immune defenses, 
and their ability to hide in the host and persist
for many years, all fit nicely with the characteristics of TSE agents. 
Highly infectious preparations with negligible PrP
contain nucleic acids of 1–5 kb, even after exhaustive nuclease digestion. 
Sedimentation as well as electron microscopic
data also reveal spherical infectious particles of 25–35 nm in diameter. 
This particle size can accommodate a viral
genome of 1–4 kb, sufficient to encode a protective nucleocapsid and/or an 
enzyme required for its replication. Host PrP
acts as a cellular facilitator for infectious particles, and ultimately 
accrues pathological amyloid features. A most
significant advance has been the development of tissue culture models that 
support the replication of many different strains
of agent and can produce high levels of infectivity. These models provide 
new ways to rapidly identify intrinsic viral and
strain-specific molecules so important for diagnosis, prevention, and 
fundamental understanding.


J. Cell. Biochem. 100:
897–915, 2007. 
 2006 Wiley-Liss, Inc.
Key words: Creutzfeldt-Jakob disease; scrapie; BSE; viral particle; latent 
infection; prion pathology


‘‘I dislike arguments of any kind. They
are always vulgar, and often convincing.’’
Oscar Wilde


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
spongiform encephalopathies (TSEs) typically
lead to neurodegeneration only after a long
asymptomatic period, with the concomitant
risk of transmission from apparently healthy
individuals [Manuelidis, 1994b]. This includes
inadvertent person-to-person transmissions
from tissue transplants, blood [Manuelidis
et al., 1978b, 1985; Tateishi, 1985], and possibly
even by dental procedures [Manuelidis,
1997]. The first positive transmissions from
circulating blood cells from both animals and
humans more than 25 years ago already
indicated that a larger population than those
expressing neurodegenerative disease might

 2006 Wiley-Liss, Inc.
Grant sponsor: NIH-NS12674; Grant sponsor: DODDAMD-
17-03-1-0360.
*Correspondence to: Laura Manuelidis, Yale Medical
School, New Haven, CT 06510.
E-mail: laura.manuelidis at yale.edu
Received 12 July 2006; Accepted 13 July 2006
DOI 10.1002/jcb.21090
silently carry these agents. Such iatrogenic
transmissions from asymptomatic individuals
are finally beginning to be appreciated with
cases of the BSE linked vCJD agent acquired by
transfusion [Peden et al., 2004]. The observation
that infected blood carries the infectious
agent to the intestinal tract, a direction opposite
to that commonly assumed, also raises the
likelihood of shedding these infectious agents
in feces, with further environmental contamination
[Radebold et al., 2001]. This is acommon
mode of spread for many viruses such as the
enteroviruses and hepatitis B.
Currently the most favored TSEhypothesis is
that the 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 resistance to limited proteolytic digestion
in a test tube assay with detergents. The
partially resistant form iscommonly designated
PrPSc, PrPCJD, PrPBSE and so forth, or PrP-res.
The latter designation is more objective because
PrP-res patterns are defined predominantly by
the host species and cell type rather than by the
infectious agent. In any case, these are all
equivalent terms used to describe partially
digested PrP bands on Western blots stained
with PrP antibodies. The corresponding pathologic
form of PrP is detected microscopically as
amyloid deposits or fibrils in tissues, cells, and
subcellular preparations. None of these molecular
and microscopic PrPs show significant
infectivity. This is a major, but not the sole
problem for the prion concept.
There are many distinct strains of TSE agents
that can be discriminated by their virulence in
different species, by their doubling times, and by
the distribution and severity of neuropathological
lesions they provoke. In contrast, vastly
different PrP-res patterns can be found in
different tissues of a single animal yet each
tissue yields only a single identical strain on
reinoculation. Thus, the agent rather than the
PrP-res pattern breeds true. Experimental
changes in PrP-res, achieved by infecting different
cell types in culture, also fail to alter agent
strain characteristics, as detailed below. There
are further caveats to the prion hypothesis at
the molecular level. For example, abnormal PrP
does not behave as a particle of homogeneous
size, unlike the infectious agent. Thus the term
‘‘prion particle’’ is misleading. Since PrP is host
encoded, it also does not activate innate immune
responses whereas the infectious agent
does. In TSEs robust host responses, as found in
other viral infections, have now been documented,
and become apparent a few weeks after
infection, well before PrP-res is detectable
[Lu et al., 2004]. The term replication, commonly
used to describe the rapid conversion of
normal PrP to PrP-res in a test tube, also bears
little, if any, resemblance to the observed 3- to
8-day doubling time of different TSE agents in
the brain [Manuelidis and Fritch, 1996].
These discrepancies bring us to the other
compelling counter argument that the causal
agent in TSEs is a virus with its own independent
genome. The epidemiology of TSE spread
indicates these infectious agents exist in the
environment and do not originate from some
spontaneous, and as yet undetectable change in
host-encoded PrP. For example, the UK BSE
agent progressively spread to countries where
contaminated materials and/or animals were
imported, such as the rest of Europe and Japan.
Environmental barriers can also prevent
spread of these agents. Australia, a country
with strict inspection and embargoes on scrapie-
infected sheep, has had no cases of scrapie, a
common infection of many European flocks of
the same genetic stock [Hunter and Cairns,
1998]. The natural route of spread within an
animal is also classically viral, with stopping
points in the lymphoreticular system [Manuelidis,
2003]. As most other viruses, TSE agents
must utilize and depend on host-cell molecules
for entry, replication, and maturation. PrP
genetic and antibody knockout studies show
that host PrP is required for at least one step in
this infectious process [Bu¨ eler et al., 1993;
Mallucci and Collinge, 2004]. Although PrP
knockout experiments supposedly ‘‘proved’’ the
prion hypothesis, temporal and other studies
had instead suggested that PrP acts as a host
susceptibility factor or ‘‘viral receptor’’ rather
than the infectious agent [Manuelidis et al.,
1987, 1988]. This logical and still viable conclusion
can also help to explain why PrP pathology
is a relatively late, and not always inevitable
response to infection.
The purpose of this review is to briefly
examine whether the claims made for prions
are justified, particularly in the light of recent
reports, and to consider whether the central
data actually indicate a conventional viral
particle that is unlikely to arise from the host
898 Manuelidis
genome. This paper also predict details of the
most probable TSE viral structure from the
evidence to date, and points out some old tricks
these viruses probably perform. These predictions
can be evaluated experimentally, and can
also lead to the identification of true infectious
candidates.
PRIONS REMAIN HYPOTHETICAL
‘‘A wealth of data’’ presumably supports the
notion that a host-protein transforms itself into
an infectious agent [Prusiner, 1998]. This
hypothesis was acclaimed by a Lasker Award
given to Stanley Prusiner ‘‘for demonstrating
how a genetic mutation can misfold ordinary
proteins, turning them into infectious agents
that mimic viruses,’’ He was subsequently
awarded a Nobel Prize in 1997 for ‘‘his discovery
of prions—a new biological principle of infection’’
and, in the words of the committee they
gave the following explication: ‘‘What is a prion?
It is a small infectious protein capable of causing
fatal dementia-like diseases in man and animals.
The most remarkable feature of prions is
that they are able to replicate themselves
without possessing a genome. . .. Until prions
were discovered, duplication without a genome
was considered impossible. . .. In 1976, when
Gajdusek received his Nobel Prize, the nature of
the infectious agent was completely unknown.
At this time, these diseases were assumed to he
caused by a new unidentified virus, termed a
slow or unconventional virus. During the 1970s,
no significant advances regarding the nature of
the agent were made, that is, not until Stanley
Prusiner took on the problem. After 10 years of
hard work he obtained a pure preparation. . ..
Strangely enough, he found that the protein was
present in equal amounts in the brains of both
diseased and healthy individuals’’ [KarolinskaInstitute,
1997].
Infectious proteins or prions thereby became
canonized, although careful review of the data
revealed many discrepancies, such as the presence
of abundant nucleic acids in ‘‘pure’’ prion
preparations, the discovery of many distinct
TSE strains during the period of ‘‘of no significant
advances,’’ and the separation of viruslike
infectious particles from abnormal PrP
[Manuelidis et al., 1995]. In addition, Koch’s
well-established principles to identify infectious
pathogens (see below) were not fulfilled. The
embrace of the prion hypothesis is apparent
from the following recent and representative
introductory statements to research papers in
the TSE field such as
(1) ‘‘The pathogenic PrPSc has the unique
property of being a self-replicating and
infectious agent that lacks nucleic acid’’
[Pan et al., 2004].
(2) ‘‘There is considerable evidence (not cited)
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’’ [Stewart
and Harris, 2005].
(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 PrPC’’ [Weissmann, 2004].
(4) ‘‘The causative agent of TSEs such as
scrapie is PrPSc, a misfolded, protease
resistant version of the normal PrPC
protein’’ [Aguzzi, 2005].
(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’’ [Collinge,
2005].
(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 the
Koch’s postulate’’ [Zou and Gambetti,
2005].
The last comment is interesting not only
because it fails to cite any of the primary data
that conflicts with the prion hypothesis, but
also because it suggests we should abandon
proven infectious principles since they do
not fit the prion hypothesis. Moreover, the
primary assumption that PrP is infectious can
be self-fulfilling and circular, especially when it
Transmissible Spongiform Encephalopathies 899
is continuously reiterated, as for example, with
the defective claim that PrP-res is proportional
to infectivity. The failure to examine nucleic
acids in infectious preparations with the tools of
modern molecular biology, such as RT-PCR and
specific labeling techniques, also diminishes
absolute statements about the lack of an agent
genome, especially when coding nucleic acids of
>1 kb have been identified in nuclease-treated
infectious preparations as detailed below. Moreover,
there are few published reports on nucleic
acid sequences in infectious TSE preparations
during the last 25 years, falsifying the claim
that ‘‘intense efforts in many laboratories’’ have
been made to identify a TSE-specific (or any
other) nucleic acid [Weissmann, 2004]. Through
a web of misleading language it has also
guided policies and conclusions that have been
detrimental for human and animal health
[Manuelidis, 2000, 2003].
Before examining the critical data that calls
into question the verity of the prion hypothesis,
Koch’s fundamental postulates, in an up to date
form to include current molecular capabilities,
are here stated. First, the infectious agent must
be invariably present in a characteristic and
constant form. Second, the pathogen must be
isolated and grown in pure culture, for example,
in an unrelated tissue culture, or preferably in a
recombinant form. Third, this cultured material
must be shown to reproduce the infectious
disease experimentally. Fourth, the identical
pathogen should be reisolated from the experimentally
inoculated subject.
PrP Infectivity Is Questionable, and
Probably Non-Existent
Table I lists the key prion claims that are
contradicted by data. Most critical are those
that fail to fulfill Koch’s postulates for an
infectious agent, for example, claims 1–4 as
well as the many features of prions that are
incompatible with those shown by the infectious
agent. First, there are numerous independent
animal model, subcellular-molecular, and tissue
culture studies demonstrating that PrP-res
is not required for infection, and it is also not
proportional to the infectious titer. Animal
transmissions from brains that lack PrP-res,
including cross-species transmissions, have
been positive [Manuelidis and Manuelidis,
1985; Lasmezas et al., 1997; Manuelidis et al.,
1997; Manuelidis and Lu, 2000; Race et al.,
2002]. The absence of detectable prions in
material that is clearly infectious goes against
Koch’s first requirement that the agent must
always be present. Amphotericin B treatment
also highlights discrepancies between PrP-res
and infectivity. This drug arrests PrP-res
accumulation in scrapie-infected hamsters
while the infectious agent continues to replicate
exponentially [Xi et al., 1992]. The reverse
pattern of dissociation has also been reported.
Infectivity declines while PrP-res accumulates
in salivary glands [Sakaguchi et al., 1993]. Such
data, under normal circumstances would be
considered convincing evidence thatPrP-res is a
secondary pathological response to infection.
Infectivity and PrP-res are also not proportional
across strains. One Creutzfeldt-Jakob disease
(CJD) agent produces 100,000-fold more infectivity
than a second CJD strain, yet these brains
show only 10-fold difference in PrP-res, a
10,000-fold discrepancy [Manuelidis, 1998].
Thus many different animal models show PrPres
is a poor marker for infectious titers, and a
lack of PrP-res does not preclude infection.
In terms of public health then, it is important
to recognize that asymptomatic but infected
individuals without detectable PrP-res may
continue to spread infection.
Marked discrepancies between PrP-res and
infectivity are also observed during subcellular
and chromatographic purifications of the infectious
agent as previously reviewed [Manuelidis,
2003]. In such studies, viruslike infectious
TABLE I. Major Claims and Assumptions of the Prion Hypothesis
Claim Experimentally Comment
(1) ‘‘PrPSc is proportional to titer,’’ and is infectious False In 
animal, culture, and molecular studies
(2) ‘‘Prion diversity is enciphered by PrPSc’’ False Agent strains 
breed true, but not PrPSc
(3) ‘‘PrP gene mutations cause spontaneous transmissible disease’’ 
False Transmissions not reproducible
(4) ‘‘Procedures that modify or hydrolyze PrPSc inactivate prions’’ 
True They also inactivate viruses
(5) ‘‘No evidence exists for a virus-like particle’’ False 25–35 
nm viruslike particles
(6) ‘‘Transmissible particles are devoid of nucleic acid’’ False 
Infectious particles with >500 nt lengths
(7) ‘‘No sign of an immune response to foreign agent’’ False Early 
innate immune responses
(8) ‘‘Accumulation of PrPSc associated with pathology’’ True PrPSc 
is a late host response
For experimental details see text. Quotes are from Prusiner, 1998, 1999.
900 Manuelidis
particles separate from abnormal but non-infectious
PrP (vide infra). This again emphasizes the
limitations of prion assays for the infectious
agent. More recently, infected cell and tissue
culture experiments have also shown a poor
correlation of PrP-res and infectivity. For example,
purified living microglia from CJD-infected
brain have no detectable prions, yet these
myeloid cells contain maximal levels of infectivity
that are equivalent to that of starting brain
[Baker et al., 2002]. Additionally, immortalized
neural cells infected with a CJD agent in vitro
can show a progressive 650-fold increase in
infectious titer while their PrP-res levels remain
constant [Arjona et al., 2004]. The most parsimonious
interpretation would be that the infectious
agent is different than abnormal PrP.
Indeed, the many PrP-res to infectivity discrepancies,
as well as the propagation of distinct
strains of TSE agents that breed true, regardless
of inconstant PrP conformations, has led to
additional speculations to preserve the prion
hypothesis. These have included the now apparently
abandoned ‘‘protein X’’ co-factor for infection,
glycosylation-induced infectious folding
patterns of PrP that determine strains, or some
yet to be discovered tertiary conformation of PrP
amyloid that is infectious [Aguzzi and Weissmann,
1997; Prusiner, 1998; Collinge, 2005].The
apparent lack of infectivity of PrP-res may also
underlie more recent descriptions of TSEs as
‘‘pseudoinfections,’’ though the epidemic spread
of BSE obviously contradicts this terminology.
It is also continues to be evident that no
recombinant PrP-res (recPrP) molecules, and
no PrP amyloid preparations, are sufficient to
transmit infection. The most recent experiments
with truncated recPrP [Legname et al.,
2004] show similarities to previous (transgenic)
Tg PrP mouse transmissions that were compromised
by laboratory contamination [Manuelidis
et al., 1997]. Rather than producing the predicted
novel properties that should be encoded
by this recPrP, serial passage of mouse brains
showed incubation times and neuropathological
characteristics only of the Chandler (RML)
scrapie strain used in that laboratory. Even
prion believers have realized contamination
as the most likely cause of these transmissions
[Couzin, 2004; Castilla et al., 2005; Nazor
et al., 2005]. Others, however, have been more
enthusiastic, with a celebratory call for ‘‘the
birth of a prion: spontaneous generation revisited’’
[Weissmann, 2005].
In a newer approach to prove PrP-res is
infectious, one serially dilutes scrapie-infected
brain homogenates with normal brain, and at
each sequential dilution sonicates and incubates
these mixtures under conditions that
promote PrP to PrP-res conversion in the
normal brain sample [Castilla et al., 2005]. A
tiny amount of infectivity was ultimately recovered
in the final ‘‘amplified’’ PrP-res samples.
These minute titers were most consistent with
carry over contamination on the sonication
probe, as found on other stainless steel probes
exposed to infected brain [Zobeley et al., 1999],
or with intrinsically imperfect brain homogenate
dilutions. Although the amyloid characteristics
and abundance of PrP-res were identical
in the starting undiluted and the final dilution
tubes, the infectivity titer decreased from about
1 million to less than 10. The reported conclusion,
that these results prove the infectivity of
PrP-res, may instead exemplify Languimer’s
description of pathological science based on
negligible small effects [Langmuir, 1989]. Other
investigators have also been unable to find
significant replication of infectivity using the
same published PrP-res conversion methodology
[Bieschke et al., 2004].
Agent Strains do not Display Any Constant
Individual PrP Conformation
The second major problem with the prion
hypothesis is that it does not account for TSE
strain diversity nor for the fidelity of these
individual strains in mammals with different
PrP sequences. Even as late as 1997, Prusiner
minimized the number of TSE strains, and
contended ‘‘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’’ [Scott et al., 1997]. The epidemic BSE
agent surely does not follow this rule. It
maintains its singular identity in every species
it has infected even though the abnormal PrP in
those species is quite variable. Representative
BSE-linked isolates from the many infected
species including primates, felines, canines,
gazella, kudu, caprines, and bovines all have
yielded the same BSE strain-specific profile in
inbred indicator mice [Bruce, 2003]. 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 same original scrapie incubation
Transmissible Spongiform Encephalopathies 901
and neuropathology characteristics [Zlotnik
and Rennie, 1965] despite major PrP differences
between sheep and mice. Conversely, most of
the different scrapie strains show no PrP-res
differences when propagated in inbred mice,
and in hamsters there is only a single TSE
strain, isolated from a mink, that provokes a
different brain PrP-res pattern than the various
other scrapie agents in this host [Scott et al.,
1997; Bartz et al., 2000]. The slow progressive
evolution of a TSE strain by repeated serial
propagation in a single species has also been
documented, and it is difficult to envision how or
why the unchanging PrP of that species would
modulate itself to produce these progressive
changes. Viruses clearly do this to enhance their
survival.
To account for strains, different conformational
folding patterns of PrP have been
hypothesized to ‘‘encode’’ or ‘‘encipher’’ strainspecific
information. Each hypothetical conformation
would propagate its own uniquely folded
form of PrP-res (Table I). Many experiments
show this suggestion to be unlikely because
disaggregating or unfolding PrP (so that it loses
its proteinase K resistance), does not reduce
infectivity and also does not alter strain characteristics
[Sklaviadis et al., 1989]. Enzymatic
removal of sugars also has no effect on either
infectivity or strain characteristics [Manuelidis
et al., 1987]. Furthermore, experimentally
changing the PrP-res and glycosylation band
pattern has no effect on a strain’s phenotype.
For example, when different TSE agents in
brain are propagated for extended passages in
monotypic cell cultures with very different PrP
and PrP-res patterns than brain, these agents
retain and reproduce their original very distinctive
strain characteristics, that is, the
infectious agent but not the PrP-res conformation
breeds true when the infected tissue
culture homogenates are reinoculated into
animals [Arjona et al., 2004]. In contrast, PrPres
characteristics are largely determined by
cell type and species and do not breed true. Thus
PrP-res folding differences are not required for
either the definition or the maintenance of
strain-specific properties. The variability of
PrP-res in different cell types bearing a single
agent strain also does not fulfill Koch’s requirement
that the causal agent, that is, PrP-res,
must have ‘‘constant properties.’’ High-resolution
structural analyses of PrP have also failed
to resolve any strain-specific features. Nevertheless,
hypothetical folding differences that
presumably encode strains continue to be illustrated
by color-coded cartoons [e.g., Weissmann,
2005].
PrP Mutations can Alter Susceptibility to Agent
Strains but do not Reproduce TSEs
Asingle amino acid mutation in a host protein
can change the ability of a virus to infect a cell
(reviewed in Manuelidis, 1994b). The classifi-
cation of rare ‘‘familial prion’’ CJD in people
with a 102 L PrP mutation assumes that this
gene mutation causes a dominantly inherited
(germline) infection rather than an increased
susceptibility to an environmental TSE pathogen
[Prusiner et al., 1995; Prusiner, 1998]. This
now textbook ‘‘familial’’ designation (Table I)
rests on data that is irreproducible. The frequent
accompanying reasoning, that inherited
infections cannot be caused by a conventional
viral structure, is also false because retroviruses,
for example, can be inherited through
the germline. Moreover, the assumption that
human TSE agents, unlike scrapie, are vertically
transmitted rather than endemic, is also
not in accord with studies of several human
agents in experimental animals. For example, a
sporadic CJD agent produced no maternal
transmission during 12 years of observation of
guinea pigs born to and housed with CJDinfected
parents [Manuelidis and Manuelidis,
1979b]. The disappearance of kuru after the
cessation of cannibalism [Gajdusek, 1977] also
makes the germline inheritance of this geographically
distinct CJD agent unlikely. Moreover,
the rare human 102 L mutation in PrP,
proposed to cause an ‘‘inherited’’ form of
transmissible CJD known as Gerstmann–
Straussler–Scheinker disease (GSS), should,
according to the prion hypothesis, define a
unique agent strain that is geographically
independent, and also cause a transmissible
disease when inserted transgenically. Instead,
geographically distinct CJD agents have been
isolated from GSS patients with this 102 Leu
PrP mutation [Nishida et al., 2005]. Furthermore,
this mutation has been incapable of
producing either PrPSc or transmissible disease
in mice with Tg copies of human 102 L PrP. The
initial transmissions reported in these Tg mice,
as well as the spongiform pathology [Hsiao
et al., 1990], were irreproducible when Tg mice
with normal rather than freakishly high copies
of this 102 L transgene were developed [Barron
902 Manuelidis
and Manson, 2003]. Additionally, neither the
high, nor the normal copy mutants produced
PrPSc, and this is an important reminder that
spongiform and vacuolar change can be caused
by factors other than a transmissible agent.
There are also many different non-infectious
and infectious causes of amyloid formation, a bpleated
conformation acquired by various types
of proteins, not just PrP. None of these other
amyloids have shown infectivity.
TSE Agents and Prions Have Different
Inactivation Profiles
Inactivation is an indirect and crude way to
define an infectious agent because very different
structures can display common inactivation
features. For example, treatments that destroy
TSE infectivity, such as extended proteolysis,
also destroy many viruses by digesting the
nucleocapsid coats that protects the viral genome
(Table I). Digestion of nucleocapsid proteins
by broad spectrum proteolytic enzymes
such as proteinase K, or disruption of viral
protein-nucleic acid cores by harsh chemicals
treatments such as 
2 M GdnHCl or boiling
in SDS, also markedly reduce viral titers
[Manuelidis et al., 1995]. Therefore, one cannot
conclude that these disruptive treatments prove
the TSEagent is a prion rather than a virus. It is
also often stated that ‘‘unusual properties’’ of
PrPSc ‘‘mimic’’ those of prions, that is, infectivity.
In fact, however, properties of the infectious
agent and PrP-res often diverge significantly.
Even Prusiner’s group has shown >99% of
infectivity can be destroyed by concentrations
of selected chemicals that have no effect on
PrP-res [Wille et al., 1996]. Additionally, the
properties of PrPSc responsible for proteinase K
resistance do not correlate with those conferring
thermostability on TSE agents [Somerville
et al., 2002]. The heat sensitivities for most
TSE strains are, moreover, quite conventional,
with substantial inactivation of several strains
between 70 and 848C. Many extraordinary
inactivation claims for TSE agents also rest on
assays for PrP-res without examination of infectivity.
Others emphasize sterilization levels of
infectivity (to 0%) that create an unbalanced
picture of a fantastical agent resistance, one
that does not reflect the more representative
99.9% of the infectious agent population. Indeed,
sterilization levels are difficult to achieve
with many conventional viruses such as hepatitis
B. Other physical differences between the
prion and the infectious agent are detailed
below.
Prion assumptions based on indirect tests and
weak correlative arguments also have ramifications
for public health. It is often assumed that
PrP-res is so resistant that it will survive the
harsh digestive conditions of the gastrointestinal
tract to invade the mucosa and infect
Peyer’s patches, a common entry pattern for a
number of sturdy viruses. Obviously, viruses
such as enteroviruses are designed to withstand
digestive conditions, but until recently no one
checked to find if prions could actually infect
animals via this likely route for the spread of
BSE. A recent study, where huge amounts of
infected brain were loaded directly in the gut,
showed that PrP-res was rapidly destroyed by
alimentary tract fluids. It was also clear that
some different infectious structure invaded to
provoke de novo accumulation of pathologic
PrP-res only after a 30-day hiatus [Jeffrey et al.,
2006]. This evidence should also bring to mind
the unexamined assumption that PrP-res
aggregates can cross the blood-brain barrier
in a naked state, or propagate a brain specific
PrP-res conformer from infected reticuloendothelial
cells that produce a different cell type
specific PrP-res conformer.
In summary, although like-to-like host PrP
sequences may be needed to convert PrP to PrPres
amyloid, this interaction and its consequences
are epiphenomena that are insuffi-
cient, and probably not required for the
replication of infectious particles. The above
data also suggests that mechanisms of amyloid
seeding or conversion are relevant for pathogenesis
in TSEs and in non-transmissible
Alzheimer’s disease, but are not essential for
encoding strain-specificities. Although pathological
PrP has proven to be a reliable surrogate
marker for TSE infection, especially during end
stage neurodegenerative disease, a lack of PrPres
does not rule out infection. This caveat needs
to be recognized in diagnostic PrP-res assays,
especially those currently used to assess infection
of livestock and human tissues that may be
transplanted, or that may inadvertently contaminate
surgical instruments [Manuelidis,
1997].
TSE VIRAL PARTICLES
While no form of PrP has proven infectivity,
there is substantial direct evidence for infectious
Transmissible Spongiform Encephalopathies 903
viral particles despite firm statements to the
contrary (Table II). To fully appreciate this, one
would have to look at data not cited in the prion
centric literature, including many scientific
publications more than 8 years old with important
primary data currently unavailable on the
Internet. Particles with the density and size of
viruses have been repeatedly found in more
purified infectious preparations, and particles
of similar diameter have also been identified in
many independent ultrastructural studies of
infected brain. Subcellular fractionation studies
show TSE infectivity concentrates with
25–30 nm particles from which PrP has been
largely removed. Moreover, disruption of the
protein-nucleic acid components of these particles
destroys 
99.5% of the starting infectivity.
Ultrastructural studies from the 1960s onwards
have also repeatedly shown what are probably
corresponding 25–35 nm particles in their
natural state in infected, but not in normal
brain cells. These viruslike structures continue
to be documented in many different TSE models
as detailed below.
Viruslike Structures Are Components of the
Most Infectious Subcellular Preparations
Between 1989 and 1995, sucrose gradient
analyses ofCJD-infected hamster brain fractions
revealed a narrow, homogeneous 120 S peak of
infectivity that separated from non-infectious
cellular material at 10 S. In contrast to this
viruslike peak of infectivity,more than75%of the
loaded abnormal PrP was found in the noninfectious
cellular protein region [Sklaviadis
et al., 1989, 1992]. In otherwords, non-denatured
PrPSc was separated from infectivity despite the
claim that it had not been and could not be
separated [Prusiner, 1999]. Subsequent experiments
by others with higher titer 263 K (237)
scrapie-infected hamster brains reproduced the
same sucrose gradient separations of infectivity
[Shaked et al., 1999], and Prusiner’s team was
even more successful in removing PrP-res from
infectious particles because no PrP was detectable
in theirmost infectious rapidly sedimenting
sucrose fractions [Riesner et al., 1996]. Sucrose
equilibriumgradients further demonstrated that
the 120 S infectious particles have a density of
1.28 g/cc. This densitymirrors conventional viral
cores constituted by nucleic acid-protein complexes.
Thus it was already likely that the
infectiousTSEparticlewas a protein-nucleic acid
complex, rather than a protein.
Micrococcal nuclease digestion removed copurifying
nucleic acids, which are easily visualized
by silver staining on gels before digestion.
However, this treatment did not alter either the
viruslike density or size of TSE infectious
particles [Sklaviadis et al., 1990, 1992; Manuelidis,
2003]. Thus neither the conventional viral
size nor the viruslike density of infectivity
could be explained by non-specific binding of
extrinsic nucleic acids. Moreover, contrary to
the claim that infectious preparations are
devoid of nucleic acid (Table I), nucleic acids
up to 5,000 nt long were extracted from
nuclease-treated particles in the 120 S infectious
peak. One of these sequences derived
from a co-sedimenting endogenous retrovirus
[Akowitz et al., 1994]. Its cognate protective
nucleocapsid protein of 60 kDa was
also visualized on 2D gels using nucleocapsid
antibodies, further confirming the extracted
retroviral nucleic acid sequence was bundled
as a true viral particle, albeit not the TSE agent.
Thus, as in many viral studies, nuclease
resistance fails to exclude a protected TSE viral
sequence in particles of the infectious 120 S
peak. Nuclease digestion instead can enhance
the purification of 25nmviruslike TSEparticles
from host nucleic acids.
The TSE infectious agent has not yet been
purified to homogeneity, and infectious preparations
often contain a large amount of
nucleic acids as well as other brain material.
Early prion claims of a nucleic acid free agent
were put forth despite the fact that the
infectious material described had enough
DNA to encode for a complete human being
[Manuelidis, 2003]. Others, including the
Weissmann and Marsh groups also found
nucleic acid sequences >300 nt long in Prusiner’s
later more purified fractions that were
claimed to have none (reviewed in Manuelidis
[2003]). Additionally, a recent publication from
Prusiner’s group reconfirms the presence of
significant quantities of nucleic acids in their
current most ‘‘purified prion’’ preparations.
Despite uncontrolled recovery and inadequate
detection methods, 10–20 ng of nucleic acid
was extracted from their 263 K scrapie
brain preparation that contained 108109
infectious doses. For a genome 1,000 nt in
length (the length of the hepatitis d virus), 1 ng
is sufficient to code for 109 infectious particles,
and thus 10–20 ng, as recovered from that
material was 10- to 200-fold greater than the
904 Manuelidis
measured infectious doses. Hence, tortuous
assumptions and calculations were used to
exclude a viral genome. No attempts were
made to examine the sequences retrieved, or
to evaluate retroviral sequences already shown
to co-purify with the TSE agent. This required
only a simple and rapid RT-PCR test with the
primers already described [Akowitz et al.,
1994]. The preparative recovery of infectivity
in their prion rich fraction was also poor, and
yielded only 0.1% of the starting brain
infectivity. In contrast, 15% of the starting
brain titer has been reproducibly recovered in
the 120 S sucrose peak [Sklaviadis et al., 1992].
Rather than examining the nature of the
nucleic acids recovered, indirect and inconclusive
radiation effects were cited to exclude a
viral genome even though several types of
conventional viral particles are highly resistant
to radiation (reviewed in Manuelidis,
1994b). Nevertheless, the claim that TSE
agents cannot be viral because they have
no genetic material has persisted, and has
prematurely narrowed the scope of TSE
research. Virtually no investigators during
the last 15 years have reported on the nucleic
acid sequences in their infectious preparations.
There is also a paucity of analysis
of proteins other than PrP. These include
nucleic acid binding and other proteins with
no affinity for PrP antibodies [Sklaviadis
et al., 1993].
Discrete 25–30 nm Particles in Infectious
Subcellular Fractions
There are additional experimental results
supporting a viral particle rather than prion
as the infectious agent in TSEs. Filtration data
claimed to show the infectious agent is a small
protein of <100 kDa with a diameter of <15 nm
have been irreproducible whereas a number of
filtration studies have found a minimal agent
size of25nm[Sklaviadis et al., 1992]. By 1992,
fast field flow sedimentation and high-pressure
liquid chromatography (HPLC), two independent
methods, revealed more exact viral dimensions.
Field flow sedimentation of the nucleasetreated
120 S infectious peak was compared
with marker spheres of different sizes. The
infectious peak continued to have a homogeneous
and narrow distribution, corresponding
to spheres of 25–30 nmin diameter, well within
the spectrum of conventional viruses. Particles
of this size were also visible ultrastructurally in
this fraction, with representative thin sections
of the 120 S peak shown in Figure 1. These
particles, as shown, did not bind PrP antibodies.
Parallel HPLC studies further showed these
infectious particles had an Mr of 106–107 Da,
anMr also compatible with viruses of 25–30 nm
in diameter. A very recent field flow analysis of
a less pure 263 K scrapie preparation has
similarly shown that infectivity co-migrateswith
round particles of 17–27 nm, although these
were interpreted as infectious PrP multimers
[Silveira et al., 2005]. Since the 25–30 nm
particles in our subcellular 120 S fractions do
not bindPrPantibodies (Fig. 1) they are probably
not prions. Instead, they resemble the viruslike
particles observed in intact infected fixed
brain. Those intracellular particles, arranged
in arrays, also lack PrP (vide infra).
Disruption of Viruslike Particles
Destroys Infectivity
A second strong reason to consider these
25 nm particles are viral rather than prionic,
is that disrupting their nucleic acid-protein
structure destroys their infectivity [Manuelidis
et al., 1995]. Brief exposure to 2 M GdnHCl
releases nucleic acids and cognate nucleic acid
binding proteins into the supernatant. Infectivity
was demonstrably destroyed because it could
not be recovered from the solubilized proteins
and nucleic acids in the supernatant, nor from
the residual particulate material. Yet these
GdnHCl-treated fractions continued to show
intact abnormal PrP multimers and aggregates,
the supposedly infectious prion form. In contrast,
low concentrations of SDS stripped off the
residual contaminating PrP from the infectious
120 S particles, but did not reduce the infectious
titer. These SDS-treated particles continued to
sediment at 100,000 g1 h, and retained their
intrinsic infectivity and intact nucleic acidprotein
complexes as assessed by 32P nucleic
acid labeling. In sum, subcellular infectious
fractions aremost consistent with a viral particle
of 25–30nmindiameter thatpossesses a genome
of >1 kb and a protective nucleocapsid-like
protein. PrP is not the major intrinsic protective
element of these particles because it can be
removed without affecting agent properties.
These findings are also not consistent with the
‘‘virino’’ hypothesis. The virino hypothesis
assumes a small subviral ‘‘informational molecule’’[
DickinsonandOutram,1988], presumably
Transmissible Spongiform Encephalopathies 905
smaller than the 1 kb hepatitis d viral genome.
This virino genome is thought to be protected by
a host protein, with PrP the main contender.
While the virino proposition is an understandableattempt
to reconcilePrPandviruslike strain
findings, it remains unsupported by any direct
experimental data, suchas an analysis of specific
small nucleic acids in infectious preparations. In
addition, structural features of a TSE virus
derived from the above subcellular, molecular,
Fig. 1. Electron micrographs of 120 S infectious material
fractionated from hamster CJD brain in thin sections. The
representative field A shows 25 nm diameter particles with
dense cores stained by uranyl acetate (arrows) but without PrP
associated gold particles (open arrows). PrP antibodies instead
bind fluffy proteinaceous material. B shows higher magnification,
and the dense spherical viruslike particles appear to be
attached to membranous (lighter grey) stems and channels. The
120 S fraction was fixed, embedded in LR white, and thin sections
were incubated with primary antibodies followed by3 nmgold
labeled secondary antibodies as described [Manuelidis et al.,
1987]. Bars are 100 nm.
906 Manuelidis
and electronmicroscopic studies also reasonably
match the features of the viruslike particles
repeatedly observed in perfusion-fixed brain
tissue of many different species infected with a
variety of TSE strains.
Viruslike Particles in Infected Brain Tissue
The first electron microscopic description of
arrays of viruslike particles of 35 nm in
diameter in infected brains dates back to a
beautifully clear and systematic study of experimental
scrapie in 1968 [David-Ferreira et al.,
1968]. This study excluded papovavirus contaminants.
Lampert identified the same ‘‘viruslike’’
particle arrays in synapses in experimental
primate CJD, and considered them to be
papovaviruses. This interpretation, and his
accompanying commentary, indicates a lack of
ambiguity about their conventional viral structure
[Lampert et al., 1971]. Similar viruslike
particles were identified in natural scrapie
[Bignami and Parry, 1971], and a number
of investigators including Narang [1974] suspected
these particles might be the TSE agent.
Uninfected controls did not display these viruslike
particles. Baringer and Prusiner [1978]
also subsequently reported similar crystalline
arrays of osmiophilic 25 nm particles in
scrapie-infected mice that ‘‘were a size consistent
with sedimentation and filtration data for
the scrapie agent.’’ These observations contradict
later claims of ‘‘the lack of evidence for a
virus of any shape’’ [Prusiner, 1999]. With our
successful passages of human CJD to guinea
pigs and hamsters by the 1970s, I looked for
similar particles in perfused and well-fixed
CJD-infected brains [Manuelidis et al., 1976,
1978a; Manuelidis and Manuelidis, 1979a], but
could not differentiate dense viruslike particles
from late neurodegenerative synaptic changes.
Since that time many additional studies
carried out by Liberski [2004] have extended
and solidified the presence of these viruslike
particles in a variety of TSE-infected brain
samples, including human and experimental
CJD, GSS, BSE linked vCJD, as well as many
different sheep derived scrapie strains propagated
in various inbred mice [Gibson and
Doughty, 1989]. These particles are often called
tubovesicular particles or structures because
they collect in an array of connected vesicular
channels in the synaptic region of neurons. In
most animal models they also appear at levels
consistent with infectious titers, and are less
numerous in lower titer brains [Jeffrey and
Fraser, 2000]. In contrast, PrP-res can be a
million fold greater than the experimentally
determined titers of ‘‘purified prion’’ preparations.
The exception to the above generalization
is the high titer 263 K scrapie-infected hamster
brain. Although 25 nm particle arrays have
been identified in this model, they are far less
numerous than expected. Whether packaging
and/or collection of these particles is different in
hamsters is not known. However, conventional
viral particles can be very difficult to find in
chronic viral brain infections, and may not
always correspond to levels of infection. In
Rabies, for example, brainstem viral particles
(Negri bodies) are obvious, but they may not be
apparent in infected cerebrum neurons actively
synthesizing abundant viral transcripts [Fu
et al., 1993].
Two additionalfindingsmake the intracellular
25–35 nm particles reasonable TSE viral candidates,
aside from their likeness to the particles
observed in high titer subcellular fractions.
First, they do not bind PrP antibodies, whereas
adjacent cellular PrP amyloid does. This suggests
their structure is independent and distinct
from PrP. However, given the popularity of the
prion hypothesis and the insistence that PrP
must be a part of the infectious agent [Prusiner,
1998, 1999], it is not surprising that these
particles were considered unlikely TSE viral
candidates [Liberski et al., 1997]. Nevertheless,
without PrP they appear remarkably similar to
the 25 nm particles found within the infectious
120 S peak. These particles also do not bind PrP
antibodies (Fig. 1) and can be stripped of residual
PrP without loss of infectivity (vide supra).
Second, tubulovesicular particles have been
identified prior to the onset ofneurodegenerative
changes, and were less numerous during this
preclinical period of lower brain infectivity, as
would be expected for a TSE virion [Jeffrey and
Fraser, 2000]. Their early appearance further
indicates they can be primary causal agents
rather than secondary pathological structures
generated by neuronal failure. Thus far these
particles have only been observed in synaptic
regions of neurons. Notably, synaptosomal fractions
made without detergents and verified for
purity by electron microscopy, have likewise
displayed the highest percent of brain infectivity
in two different CJD models [Manuelidis and
Manuelidis, 1983], further linking the 25–35 nm
synaptic particles to infectivity. If they are the
Transmissible Spongiform Encephalopathies 907
TSE virions, then they should probably be
present in high titer infected cell cultures that
lack synaptic differentiation. I have recently
identified 25–30 nm particles in arrays within
TSE-infected cells in culture, but not in mockinfected
controls. These show a strong resemblance
to those found by others in synaptic
processes of infected brains (manuscript in
preparation).
TSE Agents Follow the Route of Viruses and Evoke
Early Host-Immune Responses
This brings us to the involvement of the
reticuloendothelial immune system and its
ability to recognize and respond to the TSE
infectious agent as a foreign invader. In a classic
1967 paper, Eklund et al. [1967] showed that a
scrapie agent inoculated intramuscularly adjacent
to the sciatic nerve first replicated in
distant lymphoreticular tissues such as spleen
before invading neural tissue. The most likely
carrier of agent would be white blood cells and
this first demonstrated in 1978 in experimental
CJD [Manuelidis et al., 1978b]. 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, as well as those that evade immune
recognition. The presence of infectious TSE
agents in myeloid cells, such as migratory
macrophages, microglial cells, and dendritic
cells [Manuelidis et al., 2000; Aucouturier
et al., 2001; Baker et al., 2002] therefore
recapitulates this viral pattern of tissue preference
and progressive spread. Moreover,
infected lymphoid tissues provide a conduit
and source for latent accumulation, as well as
for subsequent reactivation and dissemination
of agent [Manuelidis, 2003]. In lymphoid tissues,
specialized follicular dendritic cells (FDC)
of the spleen display pathological PrP [Muramoto
et al., 1993] that largely accumulates at
the surface of the FDC [Manuelidis et al., 2000].
This is a place known to trap several types of
viruses including HIV. This overall pattern of
agent spread to lymphoreticular tissues with a
lag to produce appreciable titers in specific types
of target tissues is difficult to explain for a hostencoded
prion that is more widely distributed.
Although the dependence of different TSE
strains on FDC can be variable, as determined
by infection of Lymphotoxin-b and other knockout
mice with compromised genes affecting B
cell and FDC development [Klein et al., 1997;
Manuelidis et al., 2000; Shlomchik et al., 2001;
Aucouturier and Carnaud, 2002], FDC are
involved in all experimental TSE models examined
thus far, and FDC and antigen presenting
myeloid cells are built to facilitate recognition
of foreign pathogens. The presence of TSE
agents in myeloid cells also raised the issue of
an immune system response to TSE agents,
despite blanket dismissals of this possibility
[Manuelidis et al., 1997]. Therefore, we began to
look at inflammatory and innate immune
responses in experimental CJD, realizing that
a lack of neutralizing antibodies in TSEs represented
only a small facet of the complex interplay
between the host’s immune system and
foreign pathogens, particularly for latent and
persistent viruses that remain covert.
Early Innate Immune Response of Microglia
and Infected Brain
The early microglial recruitment in rat CJD
that preceded PrP-res accumulation [Manuelidis
et al., 1997] suggested microglia would be a
good place to begin analyses of the innate
immune system, particularly because these
cells with undetectable PrP-res are highly
infectious [Baker et al., 2002]. To reduce the
enormous cellular heterogeneity of brain tissue
it was advantageous to first separate and briefly
grow purified microglia. We compared cDNA of
CJD-infected versus uninfected microglia using
arrays spotted with immune pathway genes,
and identified many virus-linked inflammatory
changes. Exposing normal microglia to large
amounts of partially purified brain PrP-res did
not mimic these changes. However, the application
of standard inflammatory stimulators such
as lipopolysaccharide did activate a select group
of immune pathways with a pattern that
partially overlapped the infectious TSE pattern
[Baker and Manuelidis, 2003]. These innate
immune responses to TSE infection were activated
at substantial and significant levels (5 to
>40 normal). Additional experiments were
designed to look at interferon and interferon
pathways that are often recruited in a number
of viral infections, especially those that involve
ds RNAs. Moreover, interferon is part of a more
universal innate host-defense strategy that is
not limited to microglia, and is not activated by
abnormal PrP. TSE infection activated particular
interferon pathways, but did not lead to
interferon production. This abortive interferon
908 Manuelidis
pattern has also been observed with other latent
and persistent viruses [Baker et al., 2004].
To further test the biological significance of
the activated transcripts, these innate immune
markers were evaluated on complex brain
tissue by RT-PCR. Studies of samples collected
at 10-day intervals post-inoculation showed
many of the transcripts identified in purified
microglia were accuratemarkers of progressive
TSE brain infection. Statistically significant
and obvious elevation of 13 transcripts preceded
PrP-res accumulation by 60 days, and other
transcripts were recruited during ensuing disease
progression. Thus different groups of host
responses were specific for each particular stage
of infection. Additionally, these host responses
varied with the agent used for infection, and
could distinguish infection by two different CJD
agents in whole brain samples [Lu et al., 2004].
In sum, the host can recognize a TSE agent and
recruit its innate immune system to respond as
early as 20–30 days after inoculation, a time at
which the infectious agent begins to replicate.
In contrast, PrP-res begins to accumulate only
at 90 days post-inoculation, and is incapable of
activating the same immune pathways. Aside
from the incompatibility of these host responses
with the prion hypothesis, immune molecular
markers can provide useful diagnostic information
and suggest new therapeutic approaches to
prevent disease progression. While PrP pathology
is also clearly diagnostic at later stages of
infection, the involvement of other host molecules
will probably provide tests and insights
into the early and asymptomatic phases of
infection that are medically valuable.
Viruslike Interference and the Rapid Diagnosis
of Different TSE Strains by Co-Culture
Viral interference is a well-known phenomenon
documented for classes of viruses as
elementary as bacterial plasmids and as sophisticated
as complex poxvirus infections of mammals.
Interference occurs when infection by one
viral strain prevents superinfection by a second
related challenge agent. Although antibodies
provide the major mechanismof interference in
acute viral infections of mammals, and are the
basis for many effective vaccines, interference is
complicated and incompletely understood even
for simple bacterial plasmids [Ho et al., 2002].
The mechanisms of interference for various
mammalian viruses grown in non-lymphoid cell
cultures, where antibody responses are excluded,
can also involve several cellular pathways
and cell surface molecules [Kristal et al.,
1993]. Although understanding TSE interference
mechanisms may take years to clarify, the
capacity of specific TSE agents to interfere with
superinfection by related members of its class
strongly implicates a virus rather than a prion.
Tests of superinfection in vivo, as well as in
newly developed cell culture models, show
protection is dependent on the continued presence
of specific agent strains, and that PrP-res
is irrelevant for effective interference.
A slow and avirulent strain of CJD, typical of
sporadic CJD isolates, prevents superinfection
by a more virulent Asiatic CJD isolate in vivo
even though the slow strain provokes no
detectable PrP-res. This interference was so
dramatic [Manuelidis, 1998; Manuelidis and
Lu, 2003] reviewers had difficulty accepting the
results. The presence of the suppressed challenge
agent in these brains became evident on
further secondary passages [Manuelidis and
Lu, 2000]. Interference could also last for the
lifetime of the host. With low doses of the
protective agent, mice survived free of disease
until they began to die of old age at >650 days,
even though they were challenged with moderate
doses of the more virulent agent. Unprotected
control mice inoculated only with the
challenge agent all died 350 days earlier than
the protected mice. PrP-res was not involved in
this protection since PrP-res was undetectable
during the 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. Finally, despite the simultaneous propagation
of two distinct CJD strains in each
animal for over a year, no ‘‘chimeric’’ or intermediary
TSE strains were produced as predicted
by the prion hypothesis [Scott et al.,
1997]. Doubly infected brains gave rise only to
the two original strains, and each agent bred
true with no mixed or chimeric agent phenotype
by neuropathology and incubation time [Manuelidis
and Lu, 2000]. The prion hypothesis has
not explained these positive interference
results, shown in several experiments, and
using different routes of challenge, for example,
intracerebral and intravenous.
To find if neural cell cultures that are free of
immune system cells could support interference,
Transmissible Spongiform Encephalopathies 909
we developed a rapid and more accessible coculture
test. This format was used to evaluate a
variety of CJD and scrapie agent strains that
had similar incubation times and widespread
brain lesions, and thus could not be easily
discriminated from each other in doubly
infected mice [Nishida et al., 2005]. In vitro,
neomycin resistant target cells infected with
one agent are exposed to cells infected with a
second challenge agent, and after a few days of
co-cultivation the infected challenge cells are
removed by antibiotic selection. The pure target
cells can then be assayed for superinfection.
These experiments demonstrated that: (1)
interference between TSEagents can take place
in a simplified cell system without lymphoreticular
cells, (2) some sheep derived scrapie
strains can interfere with human-derived CJD
agents, and visa versa, and (3) interference is
dependent on the individual agent strains, but
not on the presence or absence of PrP-res. The
relationship between different scrapie and CJD
agents was surprising because it emphasized
interference was unrelated to the natural host
species for these agents. For example, two
scrapie strains with similar titers showed very
different abilities to protect cells from superinfection
by a CJD agent isolated in Japan.
A low titer CJD agent from the USA also
effectively prevented superinfection by scrapie
agents. Clearly some scrapie strains have more
in common with a human agent than with other
sheep agents, at least with respect to their
interfering and/or superinfecting capacities. All
this leads one to question any rigid distinctions
between sheep and human agents, an assumption
underlying many public health conclusions
about the inability of sheep scrapie to spread to
humans. These experiments also underscore
the potential and rarely considered possible
spread of at least some human TSE agents to
other mammals [Manuelidis and Manuelidis,
1993].
The amount and pattern of PrP-res is completely
irrelevant to interference in cell culture,
as had been shown previously in animals. PrPres
is not necessary to prevent superinfection,
since cells infected by an agent that provokes no
PrP-res were resistant to challenge by CJD and
scrapie agents. Conversely, agents that induced
very large amounts of PrP-res failed confer
protection as would be expected if different forms
of abnormal PrP-res competed for the small
amount of remaining normal host PrP. Indeed,
interference depended only on the continued
presence of the protective infectious agent. This
was demonstrated by ‘‘curing’’ target cells of
their protective infection by Pentosan polysulfate.
They then became susceptible to challenge
superinfection [Nishida et al., 2005]. This data
shows that the continued presence of the agent
itself, rather than any single host response, is the
essential determinant underlying interference.
These co-culture tests are also remarkable
because they can rapidly discriminate among
different agent strains. In the past, long and
expensive mouse incubations for 120 to more
than 350 days, in conjunction with evaluation of
subtle neuropathologic differences, were needed
to discriminate among strains, especially those
without unique PrP-res profiles. In contrast, coculture
interference experiments can resolve
strains in as little as 25 days. Additionally, if a
strain changes significantly, this may be rapidly
detected in these simplified culture interference
models. The effect of selected drugs and antisense
molecules can also be rapidly evaluated
in vitro.
While the exact mechanisms of interference
are not known they probably involve cellular
pathways used by the agent, as well as agent
specifiedmolecules, and/or variant forms of the
agent itself such as defective interfering particles.
Host molecules may limit agent entry or
replication, and/or enhance the clearance of the
challenge agent from the cell. In terms of viralbased
mechanisms, specific viral molecules
such as interfering RNA transcripts may
provide interference against invading members
with similar genomes. Strain-specific
targeting of particular intracellular compartments
and organelles may also contribute to
interference. Even in protists, intracellular
positioning has been considered to be critical
in interference or strain incompatibility, with
plasmid positions a major determinant for
successful cohabitation (reviewed in Ho et al.
[2002]). The primacy of strain characteristics in
interference further emphasizes the need to
look at these agents directly, and to characterize
their intrinsic molecules. Host responses,
including PrP changes, of course are important
for the details of pathogenesis, and when
present are very helpful for confirming infection.
But without fundamental characterization
of the TSE agent itself, the central and
probably most critical viral mechanisms will
remain obscure.
910 Manuelidis
Predictions for a TSE Virus Based on Biological,
Physical, and Molecular Data
Although the molecular nature of the infectious
TSE agent remains unknown, there is
substantial evidence for predicting that a 25–
30 nm round or dodecahedral particle with a
protected viral (nucleic acid) genome of 1–4 kb
will define specific TSE agent strains. TSE
agents depend on normal host PrP in their life
cycle, but the cumulative data indicate that PrP
is unlikely to be an intrinsic part of the
infectious particle. To summarize: (1) infectious
particles of 25–30 nm can be substantially
separated from all forms of PrP. (2) Disruption
and solubilization of the nucleic acid-protein
complexes of these particles destroys their
infectivity. (3) Viruslike structures of 25–
30 nm can be identified in nuclease-treated
120 S infectious fractions. (4) Similar viruslike
particles of 25–35 nmare observed in the brains
of various species infected with different TSE
strains, but not in any uninfected controls. (5)
PrP antibodies do not bind to either the
subcellular 120 S viruslike particles or to the
viruslike structures identified in intact cells. (6)
The ‘‘virion’’ arrays in brain increase with the
infectious titer. (7) TSE agents spread by the
same lymphoreticular pathways as viruses. (8)
They also provoke innate immune responses
diagnostic of a non-host-encoded environmental
pathogen. (9) TSE agents display individual
strain characteristics and viruslike interfering
capacities that are independent of pathological
PrP-res expression.
These key viral features are summarized in
Table II. The predicted genome size derives
from the physical and molecular data. This
genome size has a potential to encode a
nucleocapsid protein for protection, and/or an
enzyme necessary for replication of the viral
genome. Thus one may uncover a viral nucleic
acid binding protein, or a sequence motif
involved in nucleic acid replication. Predicting
the features of a TSE virus can be helpful to
future research since it provides specific guidelines
for judging if an isolate or structure
obtained is a reasonable infectious candidate.
It is also important to recognize some potential
pitfalls in looking for a non-host-nucleic
acid sequence in TSEs. If these viruses are
more common, or commensal, as previously
suggested (e.g., Manuelidis and Manuelidis,
1979a; Manuelidis, 1994a; Manuelidis and Lu,
2003, and Table II legend) then they may also be
present in ‘‘normal’’ brains at some low level.
There are a number of typically non-pathogenic
commensal viruses that persist in the host for a
lifetime, such as the JC papova virus that has
been identified in the brains of over 50% of the
asymptomatic human population [White et al.,
1992]. This problem, in part, may be overcome
by sequencing various agent strains in vivo and
in vitro and in different species and tissues. The
counter argument can be made, that different
agents in various models will not be sufficiently
similar to identify a shared or common viral
sequence. However, the stability of all these
agents and their ability to breed true, indicate
a highly conserved and invariant genome.
Examples include the unchanging characteristics
of the BSE agent after passage through
widely divergent species, the maintenance of
distinct CJD agent strains despite passage
in the same monotypic cell line [Arjona
et al., 2004], the stability of sheep scrapie for
TABLE II. Key Biologic, Physical, and Molecular Features of TSE Agents From 
the
Cumulative Experimental Data
Viral properties Comment
Preference for lymphoid tissues and brain Typical hematogenous viral spread 
to specific tissues
Pathogenic and non-pathogenic variant strains Virulent versus predominantly 
asymptomatic and species limited
TSE specific members of class Need host PrP
25–30 nm diameter infectious particle without PrP From field flow 
fractionation and morphology
Mr of 106–107 Da By HPLC of nuclease-treated 120 S sucrose peak
Can cantain a foreign viral genome of 1–4 kb May encode replication and/or 
protective capsid protein
The prediction that the TSE agentwill have a viral genome of 1–4 kb is 
based on the empirical physical properties of infectious particles,
the presence of nucleic acids of these lengths that are resistant to 
nuclease digestion, and the requirement for specifying individual TSE
strains. Normal host PrP is a known required susceptibility factor as shown 
by PrP knockout studies [Bu¨ eler et al., 1993], but there are
also other tissue and cell type specific factors, unrelated to PrP, that 
determine susceptibility. The extraordinarily long latency and
asymptomatic persistence of most TSE infections in humans, as in kuru 
[Gajdusek, 1977], the appearance of CJD as long as 30 years
after exposure to contaminated growth hormone in only some of the exposed 
people [Manuelidis, 1994b], and the low incidence of late
onset sporadic CJD are also some of the reasons to consider relatively 
avirulent or non-pathogenic TSE members that may be
commensal, or morecommon than realized.Non-specific stress, as well as other 
infections and diseases, or even aging itself [Manuelidis,
1994a] may allow non-pathogenic commensal TSE agents to recrudesce from a 
latent carrier state to one causing disease.
Transmissible Spongiform Encephalopathies 911
centuries, and the slow evolution of these agents
in cross-species infection [Manuelidis et al.,
1997]. The overall similarity of different TSE
strains with respect to tissue preference, spongiform
lesions, and PrP pathology also suggest
they will have a common consensus or shared
sequence.
New Impartial and Rapid Tests That can Prove if a
Virus or a Prion Is the Infectious Agent
Fewscientists have attempted to characterize
the infectious agent, whereas most have
chosen instead to follow all the twists and turns
of pathological PrP, and this in part has been
due to the length and expense of the TSE
infectious assay. With the new infected cell
culture models that are capable of supporting
high levels of agent replication and a variety of
agents strains, as well as the development of
rapid tissue culture assays to discriminate
among these strains [Arjona et al., 2004;
Nishida et al., 2005], the impediments of in vivo
infectious assays can be minimized. Cell
cultures that have been shown to support
levels of infectivity that are comparable to
brain provide new opportunities to purify and
rapidly evaluate the essential infectious particle
from non-degenerating cells. Because cultured
monotypic cells do not carry complex
brain tissue elements such as myelin, collagen,
glial fibers, and vascular material, it is likely
that agent purification will be simplified. Molecules
in more purified infectious particles from
these cells should become obvious above a
simplified background of material from one cell
type.
Susceptible tissue culture cells can also
provide the essential assay system for rapidly
screening infectious fractions. Although it
remains to be seen if cells in culture are as
quantitatively accurate as serial dilution endpoints
determined in animals, they can be
useful for assessing the relative infectivity of
subcellular fractions within 30 days (unpublished
observations). Thus one should be able to
trace if a newly identified candidate viral
molecule corresponds to, and predicts, infectious
titers in increasingly purified preparations.
Ultimately agent specific molecules
should become apparent. If a TSE specific
nucleic acid sequence is identified, then the
wholeTSEfield will be opened on a fundamental
as well as an exquisitely sensitive diagnostic
level, given the power of modern molecular
techniques such as PCR. If a nucleocapsid-like
protein is found, antibody responses of the host
may be enhanced for prevention of infection by
vaccination. The failure to find neutralizing
antibodies in TSEs does not rule out the
elaboration of antibodies against a TSE virion,
since neutralization is a relatively insensitive
test for covert and evasive viruses.
The development of TSE susceptible cells also
trumps all other methods of objectively evaluating
whether the infectious agent is a prion or a
virus by Koch’s criteria. This involves more than
just the greater simplicity and rapid readout of
cell cultures. Various purified molecules can be
delivered much more efficiently to cells in
culture than in brain. More than 95% of the
inoculum is lost from brain in the first few hours
of infection [Manuelidis and Fritch, 1996]
whereas cells in culture can be exposed to high
concentrations of agent for extended periods.
The development of susceptible cells also provides
a chance to evaluate small amounts of
pure molecules that would be degraded shortly
after inoculation in an animal. Viral candidate
molecules include naked nucleic acid sequences,
as in an engineered plasmid that may encode
sufficient information for TSE particle synthesis
in the cell. Recombinant PrPs in various
states that have not produced clear infection by
brain inoculation can also be rapidly tested to
see if they can reproduce infectivity in cell
cultures. These cell-based infectivity assays are
more biologically meaningful than non-physiological
test tube manipulations to produce noninfectious
PrP-res. Finally, cells that show the
hallmarks of infection after delivery of candidate
agent molecules can be inoculated as back
into animals. This would be the ultimate proof
that the molecule(s) identified fulfill Koch’s
postulates. In animals, these infected cells
should produce both transmissible infection
and a specific disease phenotype [Arjona et al.,
2004]. Modification of these molecules, as by
targeted mutagenesis of nucleic acids should
also clarify the origin of different strains. This
strategy seems the most objective way to
systematically clarify intrinsic agent molecules,
viral, prion, or otherwise.
CONCLUDING REMARKS
Cell culture models provide an exciting new
opportunity to perform critical and fundamental
experiments that can elucidate the intrinsic
912 Manuelidis
molecular components of infectious TSE agents.
Candidate viral or prion molecules can be
evaluated for their infectivity and strain determining
properties. The development of in vitro
cell assays should please everyone, including
those who favor prion, viral, or other hypotheses,
because these assays can be used to
objectively and efficiently test many different
isolates and preparations according to Koch’s
postulates.
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