Cells infected with scrapie and CJD produce 25-nm virus-like

[flatfish]

Cells infected with scrapie and CJD produce 25-nm virus-like
Thu Feb 1, 2007 10:17
71.248.138.100


Subject: Cells infected with scrapie and Creutzfeldt–Jakob disease agents produce intracellular 25-nm virus-like particles
Date: January 31, 2007 at 10:28 am PST
Cells infected with scrapie and Creutzfeldt–Jakob

disease agents produce intracellular 25-nm

virus-like particles


Laura Manuelidis*, Zhoa-Xue Yu, Nuria Banquero, 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 infectionassociated

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.



snip.



In summation, all of this data provides a clear, consistent,

substantive, and logical alternative to the accepted prion hypothesis.

The causative TSE agent is most consistent with an

exogenous 25-nm virion without intrinsic host PrP. The stimulation

of host innate immune responses by these agents, a

complex set of molecular reactions that precedes the elaboration

of pathologic PrP (9) and one that is not provoked by PrP-res

itself (25), also point to a foreign pathogen rather than some

unpredictably spontaneous mutation in the host’s PrP without

cause. The presence of these particles in many different species

infected with a wide variety of TSE strains is in accord with

Koch’s first requirement (1). It is also improbable that an

identical virus-like structure would be a contaminant or a

secondary coincidental feature of all these different TSE models.

Nevertheless, a more detailed molecular analysis of these

particles will be required to substantiate their causal nature.

Purification of these
25-nm particles from productive tissue

cultures should be informative if the essential infectivity assays

are performed systematically with parallel ultrastructural and

molecular analyses. Animal titrations of infectivity are expensive

and prolonged. However, sustained and reproducible infection

of indicator GT cells by a variety of TSE agents already has

shown that they can rapidly authenticate the presence of agent

in disrupted samples as well as in living cells (4, 17). GT cells also

may be used for testing infectivity of viral nucleic acids as well

as PrP conformers. Rapid assays of infectivity in culture should

facilitate the isolation of infectious particles from host components,

and treatments that modify the production of these

particles in culture may resolve further the infectious structure

from the pathological disease processes it initiates.

Materials and Methods......snip......end........TSS


http://www.pnas.org/cgi/content/abstract/0610999104v1



Transmission studies do not lie, and neither do the threshold of destruction i.e. 600 degrees c. does not disprove what has already been proven about amplifications and transmissions of this agent. ...TSS


1: J Infect Dis 1980 Aug;142(2):205-8


Oral transmission of kuru, Creutzfeldt-Jakob disease, and scrapie to nonhuman primates.

Gibbs CJ Jr, Amyx HL, Bacote A, Masters CL, Gajdusek DC.

Kuru and Creutzfeldt-Jakob disease of humans and scrapie disease of sheep and goats were transmitted to squirrel monkeys (Saimiri sciureus) that were exposed to the infectious agents only by their nonforced consumption of known infectious tissues. The asymptomatic incubation period in the one monkey exposed to the virus of kuru was 36 months; that in the two monkeys exposed to the virus of Creutzfeldt-Jakob disease was 23 and 27 months, respectively; and that in the two monkeys exposed to the virus of scrapie was 25 and 32 months, respectively. Careful physical examination of the buccal cavities of all of the monkeys failed to reveal signs or oral lesions. One additional monkey similarly exposed to kuru has remained asymptomatic during the 39 months that it has been under observation.

PMID: 6997404
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6997404&dopt=Abstract

1: J Neurol Neurosurg Psychiatry 1994 Jun;57(6):757-8


Transmission of Creutzfeldt-Jakob disease to a chimpanzee by electrodes contaminated during neurosurgery.

Gibbs CJ Jr, Asher DM, Kobrine A, Amyx HL, Sulima MP, Gajdusek DC.

Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.

Stereotactic multicontact electrodes used to probe the cerebral cortex of a middle aged woman with progressive dementia were previously implicated in the accidental transmission of Creutzfeldt-Jakob disease (CJD) to two younger patients. The diagnoses of CJD have been confirmed for all three cases. More than two years after their last use in humans, after three cleanings and repeated sterilisation in ethanol and formaldehyde vapour, the electrodes were implanted in the cortex of a chimpanzee. Eighteen months later the animal became ill with CJD. This finding serves to re-emphasise the potential danger posed by reuse of instruments contaminated with the agents of spongiform encephalopathies, even after scrupulous attempts to clean them.

PMID: 8006664 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8006664&dopt=Abstract



New studies on the heat resistance of
hamster-adapted scrapie agent: Threshold
survival after ashing at 600°C suggests an
inorganic template of replication

Paul Brown*,, Edward H. Rau, Bruce K. Johnson*, Alfred E. Bacote*, Clarence J. Gibbs Jr.*, and D. Carleton Gajdusek§

http://www.pnas.org/cgi/content/full/97/7/3418


TSS

[flatfish]

Published: Wednesday, February 7, 2007


Yale M.D. makes leap in mad cow research

Misha Mihailova

Staff Reporter


Though challenging the accepted theory of the causes of mad cow disease may
seem like madness itself, a team of researchers from the Yale School of
Medicine has announced potentially groundbreaking findings concerning the
origins of the disease.

Yale School of Medicine professor Dr. Laura Manuelidis, the head of
neuropathology at the school, and her team of researchers recently published
a report in the Proceedings of the National Academy of Sciences asserting
that a virus, rather than prion proteins, is the cause of mad cow disease in
animals and Creutzfeldt-Jakob disease in humans. These spongiform
encephalopathies of the brain have traditionally been thought to be caused
by prions — abnormal proteins that convert healthy proteins to the disease
state. But the new study suggests prions may simply be part of the late
stages of the diseases, not part of the causes.

Transmissible spongiform encephalopathies affect the brains and nervous
systems of victims, including sheep, deer and humans. The term ”spongiform”
comes from the fact that the infection causes neurons to die, leaving tiny
holes in the brain so that it eventually resembles a sponge. Humans
afflicted with the incurable degenerative disease show memory and
personality changes and sometimes problems with movement. Mad cow, the
cattle version of the disease, has achieved infamy because the infectious
agent in cows appears to be the cause of vCJD, a variant form of CJD in
humans.

The research team’s goal was to try to identify viral particles in infected
cells. They infected cell lines with either scrapie (a sheep disease related
to mad cow) or CJD agents and found virus-like particles that did not
contain prion protein. An abundance of these particles was related to high
levels of infectivity, which was not true of the presence of prion proteins.

“People hypothesize that prion proteins are infectious, but they’re probably
part of the disease, not the infectious agent itself,” Manuelidis said.

The virus-like particles had been found by other researchers but were
largely ignored. They were first identified in 1968 in synaptic regions of
scrapie-infected brain and later found in many other animals with different
TSEs. But Manuelidis said that researchers apparently forgot about them once
the prion hypothesis became dominant.

“I had totally forgotten about them, too,” she said. “But after we found the
25-nanometer particles, I went home and remembered I’ve seen these before. I
went back to the old journals from the ’70s and there they were.”

Manuelidis said part of the reason for the neglect of the virus-like
particles is that in previous studies, they were always found in
degenerating brain tissue, so it was impossible for scientists to draw
accurate conclusions about them. What allowed her to collect relevant
experimental data was the fact that her team observed the particles in
highly infectious tissue culture cells.

“We saw them in tissue culture cells that weren’t degenerating,” she said.
“The infected cells were as healthy as the uninfected cells that had no
25-nm virus-like particles.”

The study is a work in progress, and the researchers want to conduct more
experiments with tissue cultures to gather further evidence and learn more
about the particles. Manuelidis said that since it is easier to work with a
simplified cell system than with infected animals whose brains are
degenerating, tissue culture experiments can be used to identify essential
features of the infectious agent and clarify the way in which the particles
invade cells. Her team will use the tissue cultures to purify the virus-like
particles more completely.

“In the future, we will try to isolate the particles from tissue cultures
and characterize what is in them,” she said.

The team’s specific viral particle hypothesis suggests new avenues for
treatment and vaccines, Manuelidis said. If they are successful in rapidly
measuring infectivity in tissue culture, they will be able to gain better
understanding of possible remedies. She even pointed out that prion
proteins, while not the infectious agent, are probably essential receptors
for replication and growth of the TSE virus.

”The infectious agent needs prion proteins to grow,” Manuelidis said. “This
means targeting the prion protein may also be a useful therapeutic step.”

Researchers at other universities pointed out that the Yale study does not
definitely prove the viral hypothesis, nor does it fully disprove the prion
hypothesis.

Tricia Serio, an assistant professor of molecular biology, cell biology and
biochemistry at Brown University, said that while the research is
intriguing, the viral hypothesis still needs to be directly proven. She also
pointed out that there are many examples of protein-based phenotypes, like
the one described in the prion hypothesis.

“For example, we study prions in yeast, and work from the Weissman, King,
Liebman, Saupe and Wickner labs has shown that the transfer of recombinant
protein produced in bacteria is sufficient to induce a heritable phenotype
in yeast,” she said. ”This is direct proof of a prion mechanism for the
yeast traits.”

Surachai Supattapone, associate professor of biochemistry and medicine at
Dartmouth University, said that the next challenge for Dr. Manuelidis’ group
will be to isolate and identify a specific virus that can cause
transmissible spongiform encephalopathy. He said, however, that there is
still work ahead for prion protein researchers.

“Proponents of the ‘protein only’ hypothesis — which is also not proven —
will need to demonstrate that purified prion proteins alone can fulfill Koch
’s postulates [a set of criteria for establishing a causal relationship
between an infectious agent and a disease] to prove their alternative
claim,” he said.


http://www.yaledailynews.com/articles/view/19788


TSS