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From: TSS ()
Subject: Disease-specific particles without prion protein in prion diseases – phenomenon or epiphenomenon?
Date: July 13, 2007 at 9:49 am PST

Disease-specific particles without prion protein in prion diseases –
phenomenon or epiphenomenon?

P. P. Liberski**Department of Molecular Pathology and Neuropathology,
Medical University of Lodz, Lodz, Poland; and Pawel P. Liberski, Department
of Molecular Pathology and Neuropathology, Medical University of Lodz,
Czechoslowacka Street 8/10, PL 92-216 Lodz, Poland. Tel: +42-679-14-77; Fax:
+42-679-14-77; E-mail: and P. Brown††Refired,
Bethesda, Maryland, USA *Department of Molecular Pathology and
Neuropathology, Medical University of Lodz, Lodz, Poland; and †Refired,
Bethesda, Maryland, USA
Pawel P. Liberski, Department of Molecular Pathology and Neuropathology,
Medical University of Lodz, Czechoslowacka Street 8/10, PL 92-216 Lodz,
Poland. Tel: +42-679-14-77; Fax: +42-679-14-77; E-mail:
P. P. Liberski and P. Brown (2007) Neuropathology and Applied Neurobiology
33, 395–397

Disease-specific particles without prion protein in prion diseases –
phenomenon or epiphenomenon?


The search for the cause of transmissible spongiform encephalopathies (TSEs)
has a long and tortuous history. In a recent paper, 25-nm virus-like
particles were identified that were consistently observed in cell cultures
infected with Creutzfeldt-Jakob disease (CJD) and scrapie; they are similar
to, or even identical with, the virus-like tubulovesicular structures (TVS)
found in experimental scrapie as early as in 1968, and subsequently in all
naturally occurring and experimentally induced TSEs. These particles have
been viewed with caution by the scientific community because of the
unverified or uninterpretable record of virus-like structures reported over
the years in TSEs. TVS are spherical or tubular particles of approximate
diameter 25–37 nm. They are smaller than synaptic vesicles, but larger than
many particulate structures of the central nervous system, such as glycogen
granules. Their electron density is higher compared with synaptic vesicles,
and in experimental murine scrapie, they form paracrystalline arrays. None
of these observations distinguish between TVS as an entity critical to the
infectious process, or as a highly specific ultrastructural epiphenomenon,
but their consistent presence in all TSEs demands further research.

Confusious is confused again ???

An epiphenomenon is a secondary phenomenon that occurs alongside a primary

Often, a causal relationship between the phenomena is implied: the
epiphenomenon is a consequence of the primary phenomenon. In medicine, this
relationship is typically not implied: an epiphenomenon may occur
independently, and is merely called an epiphenomenon because it is not the
primary phenomenon under study. (A side-effect is a specific kind of
epiphenomenon that does occur as a consequence.)

In philosophy of mind, epiphenomenalism is the view that mental phenomena
are caused by physical phenomena, and cannot cause anything themselves. It
was probably first mentioned by T. H. Huxley in 1874.

Marvelous that the authors ignore the fact that Manuelidis et al first
reported dense 25nm diameter particles
(by field flow fractionation in 1992) in highly infectious brain fractions
with little PrP. Unlike PrP, these viruslike
particles have not been found in uninfected brains. Of course they are
important to characterize.
Though the authors seem to dismiss them as epiphenomena. Could the dense
25nm particles be the Phenomenon,
with the PrP the Epiphenomenon ? which came first, the horse or the cart ???
TSS et al ;-)

Cells infected with scrapie and Creutzfeldt–Jakob disease agents produce
intracellular 25-nm virus-like particles

Laura Manuelidis*, Zhoa-Xue Yu, Nuria Barquero, and Brian Mullins

Yale Medical School, 333 Cedar Street, New Haven, CT 06510

Communicated by Sheldon Penman, Massachusetts Institute of Technology,
Cambridge, MA, December 11, 2006 (received for review October 10, 2006)

We had repeatedly found 25-nm-diameter virus-like particles in highly
infectious brain fractions with little prion protein (PrP), and therefore we
searched for similar virus-like particles in situ in infected cell lines
with high titers. Neuroblastoma cells infected with the 22L strain of
scrapie as well as hypothalamic GT cells infected with the FU
Creutzfeldt–Jakob disease agent, but not parallel mock controls, displayed
dense 25-nm virus-like particles in orthogonal arrays. These particles had
no relation to abnormal PrP amyloid in situ, nor were they labeled by PrP
antibodies that faithfully recognized rough endoplasmic reticulum membranes
and amyloid fibrils, the predicted sites of normal and pathological
intracellular PrP. Additionally, phorbol ester stimulated the production of
abnormal PrP gel bands by >5-fold in infected N2a + 22L cells, yet this did
not increase either the number of virus-like arrays or the infectious titer
of these cells. Thus, the 25-nm infection-associated particles could not be
prions. Synaptic differentiation and neurodegeneration, as well as
retroviruses that populate the rough endoplasmic reticulum of neuroblastoma
cells, were not required for particle production. The 25-nm particle arrays
in cultured cells strongly resembled those first described in 1968 in
synaptic regions of scrapie-infected brain and subsequently identified in
many natural and experimental TSEs. The high infectivity of comparable,
isolated virus-like particles that show no intrinsic PrP by antibody
labeling, combined with their loss of infectivity when nucleic acid–protein
complexes are disrupted, make it likely that these 25-nm particles are the
causal TSE virions that induce late-stage PrP brain pathology.

infection | neuroectodermal cultures | virion ultrastructure | prion amyloid
| retrovirus

Author contributions: L.M. designed research; L.M., Z.-X.Y., N.B., and B.M.
performed research; L.M., Z.-X.Y., and N.B. analyzed data; and L.M. wrote
the paper.
The authors declare no conflict of interest.

*To whom correspondence should be addressed. E-mail:

© 2007 by The National Academy of Sciences of the USA

A 25 nm virion is the likely cause of transmissible spongiform
Laura Manuelidis *
Yale Medical School, New Haven, Connecticut 06510

email: Laura Manuelidis (

*Correspondence to Laura Manuelidis, Yale Medical School, New Haven, CT

Funded by:

Creutzfeldt-Jakob disease • scrapie • BSE • viral particle • latent
infection • prion pathology

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.

Received: 12 July 2006; Accepted: 13 July 2006
Digital Object Identifier (DOI)

Copyright © 1992 Published by Elsevier Science B.V.

Analysis of Creutzfeldt-Jakob disease infectious fractions by gel permeation
chromatography and sedimentation field flow fractionation

T. Sklaviadis1, R. Dreyer2 and Laura Manuelidis
Yale University, School of Medicine, New Hauen, CT 06510, USA
Received 17 June 1992; revised 3 September 1992; accepted 8 September 1992.
Available online 5 November 2002.


Gel permeation chromatography and sedimentation field flow fractionation
(SF3) were used to further analyze highly infectious fractions from
Creutzfeldt-Jakob disease (CJD) infected hamster brain. These analyses
defined the relative molecular mass and physical size of the
Creutzfeldt-Jakob disease (CJD) agent with greater precision than previously
possible. Highly purified disaggregated fractions yielded single,
homogeneous Gaussian peaks with both methods. The relevant analytical peaks
contained protein-nucleic acid complexes with an Mrr of ˜ 1.5 × 107 daltons
and a mean radius of ˜ 30 nm. The experimental evidence further solidifies
the concept of an infectious agent that resembles a viral core rather than a
simple protein.

Author Keywords: Scrapie; Creutzfeldt-Jakob Disease; Gel permeation
chromatography; Sedimentation field flow fractionation; Agent radius;
Molecular mass

1 Present address: Aristotle University of Thessaloniki, School of Health
Sciences, Department of Pharmaceutical Sciences, Thessaloniki 54006, Greece.

2 Present address: Miles Laboratories MRC, 400 Morgan Lane, West Haven, CT
06516, USA.

Correspondence to: L. Manuelidis, Yale University, School of Medicine, 310
Cedar St., , New Haven, CT 06510, , USA.

1. Sklaviadis, T., Manuelidis, L. and Manuelidis E.E. (1986).
Characterization of major peptides in Creutzfeldt-Jakob disease and scrapie.
Proc. Natl. Acad. Sci. USA 83:6146-6150.

2. Manuelidis, L., Sklaviadis, T. and Manuelidis, E.E. (1987). Evidence
suggesting that PrP is not the infectious agent in Creutzfeldt-Jakob
disease. EMBO J 6:341-347.

3. Manuelidis, L., Tesin, D., Sklaviadis, T. and Manuelidis, E.E. (1987).
Astrocyte gene expression in Creutzfeldt-Jakob disease. Proc. Natl. Acad.
Sci. USA 84:5937-5941.

4. Manuelidis, L., Sklaviadis, T. and Manuelidis, E.E. (1987). On the origin
and significance of scrapie associated fibrils. In: Court, L., Dormont, D.,
Brown, P., Kingbury, D.T. (eds) 2nd International Symposium of
Unconventional Virus Diseases of the Central Nervous System. Masson, Paris,
pp 489-507.

5. Sklaviadis, T.K., Manuelidis, L. and Manuelidis, E.E. (1989). Physical
properties of the Creutzfeldt-Jakob disease agent. J. Virol. 63:1212-1222.

6. Murdoch, G.H., Sklaviadis, T., Manuelidis, E.E., Manuelidis, L. (1990).
Potential retroviral RNAs in Creutzeldt-Jakob disease. J. Virol.

7. Sklaviadis, T., Akowitz, A., Manuelidis, E.E., Manuelidis, L. (1990).
Nuclease treatment results in high specific purification of
Creutzfeldt-Jakob disease infectivity with a density characteristic of
nucleic acid-protein complexes. Arch. Virol. 112: 215-229.

8. Akowitz, A., Sklaviadis, T., Manuelidis, E.E., Manuelidis, L. (1990).
Nuclease-resistant polyadenylated RNAs of significant size are detected by
PCR in highly purified Creutzfeldt-Jakob disease preparations. Microbial
Pathogenesis 9:33-45.

9. Sklaviadis, T., Dreyer, R., Manuelidis, L.(1992) Analysis of
Creutzfedt-Jakob disease infectious fractions by gel permeation
chromatography and sedimentation field flow fractionation. Virus Research
26, 241-254.

10. Sklaviadis, T., Akowitz, A., Manuelidis, E.E., Manuelidis, L. (1993)
Nucleic acid binding proteins in highly purified Creutzfeldt-Jakob disease
preparations. Proc. Natl. Acad. Sci. USA, 90: 5713-5717.

11. Vizirianakis, J.S., Tsiftsoglou A., S., Sklaviadis, T. Developments in
slow virus research: Implications of PrP protein involvement in cell growth
and differentiation (1993), Bovine Spongiform Encephalopathy. Where are we
now? Commision of the European Communities, 14-15 September 1993, Brussels

12. Akowitz, A., Sklaviadis, T., Manuelidis, L. (1994) Endogenous viral
complexes with long RNA cosediment with the agent of Creutzfeldt-Jakob
Disease. Nucl. Acids. Res., 22: 1101-1107.

13. Manuelidis, T. Sklaviadis, A. Akowitz, W. Fritch (1995) Viral particles
are required for infection in neurodegenerative Creutzfeldt-Jakob disease
Proc. Natl. Acad. Sci. USA , 92: 5124-5128.

14. Papaconstantinou, E., Karakiulakis, G., Roth, M., Dawson,M., Keyes, P.,
Papadopoulos,O.,Sklaviadis,T. (1999) Glycan alterations associated with
bovine spongiform encephalopathy (Accepted in archives of biochemistry and

15. S.Verghese-Nikolakaki, H. Mihaloudi, M. Polymenidou, M. Groschup, G.
Papadopoulos, T. Sklaviadis (1999) Expression of the prion protein in the
rat forebrain-, an immunohistochemical study. Neuroscience letters 272 9-12

16. S. Leontidis, V. Psychas, S. Argyroudis, A. Giannti-Stefanou, E.
Paschaleri-Papadopoulou, T. Manousis, T. Sklaviadis (1999) An overeleven
year survey of neurologic diseases of ruminats with special reference to
transmissible spongiform encephalopathies (TSEs) in Greece (Accepted for
publication Journal of Veterinary Medicine)

17. T. Manousis, M. Sachsamanoglou, P. Toumazos, S. Vergehese-Nikolakaki, O.
Papadopoulos, T. Sklaviadis (1999) Western blot detection of PrP Sc in
Cyprus sheep with natural scrapie (Accepted for publication The Veterinary

18. Vassilikioti ,O. Papadopoulos,T. Sklaviadis (1999) Reverse transriptase
activity in BSE infectious fractions. (Submitted)

19. Sachsamanoglou, M., Lolis, E., Sklaviadis, T. (1999) Thermostable and
chemically induced intermediate conformations of human PrP protein

20. T. Manousis, P. Keyes, I. Dexter, R. Green, M. Sachsananoglou, M.
Dawson, S. verghese-Nikolakaki, O. Papadopoulos, T. Sklaviadis (1999)
Characterization of the murine BSE infectious agent (Submitted Journal of
General Virology)


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