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Major CJD Discoveries

Experimental transmission of distinct human CJD agents

  • Transmissions of human sporadic CJD (sCJD) to small laboratory animals (guinea pigs, hamsters, mice) (23-25). Previously, it was believed that human CJD and kuru agents could only infect primates. Experimental rodents reproduced typical TSE lesions of humans, and the sCJD agent was unlike any sheep scrapie strain.

  • Infection is first spread by white blood cells to the lymphoreticular tissues, a typical viral route (26,27).

  • Positive CJD transmission from cornea inoculated into the eye (28). This study also underscored the potential for inadvertent iatrogenic infections.

  • No maternal transmission from infected parent guinea pigs for 12 years, i.e., agent is not germline (29)

  • sCJD agent and Asiatic CJD agents are markedly different, implicating environment-specific source (30)

  • TSE agents show conserved identities after passage through different species, and are not host-encoded (30)

  • Cultures derived from infected animals, or cells exposed to infectious material, can become transformed, and can also cause huge tumors in nude mice (31-33).

HUMAN TSE STRAINs: Sporadic sCJD in normal mice elicits only small localized PrP deposits (red) in the thalamus after a very long incubation of >420 days (left). In contrast, Asiatic isolates such as FU-CJD (right) rapidly induce widespread PrP deposits.

CJD agents transmit with viral biology & induce PrP amyloid

SAF in CJD: Nature 1983 (SAF later renamed prion rods)
  • First demonstration of scrapie associated amyloid fibrils (SAF) with PrP-res in sCJD (34-36)

  • PrP is a glycoprotein of 34kd, and deglycosylation does not alter infectivity or agent strain characteristics (36,37)

  • Many experiments show that the amount of PrP-res (the “infectious prion”) does not correlate with the amount (titer) of any form of PrP.

  • CJD infected microglia with minute amounts of PrP and with no detectable PrP-res have very high infectivity titers (41).
  • In animals, TSE agents replicate exponentially and continuously. Only after a long silent phase of agent doubling does the agent induce PrP-res, clinical signs, and spongiform neurodegeneration (38). This indicates that PrP-res is a pathological response to infection, not the causal infectious agent

  • CJD agents, as other foreign viral agents, can induce innate immune responses early after infection. In contrast, PrP-res, a host-encoded molecule that is not foreign, does not induce these responses (39,40).

  • TSE agents show virus-like INTEFERENCE in mice. Silent infection with the sCJD agent can prevent superinfection by the more virulent Asiatic (FU-CJD) agent, even though sCJD provokes no visible PrP-res.

  • No intermediate mixed or “chimeric” PrP bands are formed during infections with 2 agent strains (42,43), i.e., different TSE agents can co-exist and maintain their identity in an animal with one PrP band pattern.

  • sCJD infected cells in vitro, without detectable PrP-res, can also prevent infection by other TSE strains. This underscores the broader property of TSE interference that is common to viruses but not proteins (44).

  • Altering PrP bands by growth in different cell types does not alter agent properties (45).

  • On the basis of successful transmissions it was predicted to the MRC that the mad-cow (UK-BSE) agent could transmit to humans, regardless of species PrP differences between humans and cows (46). We developed rapid sterilization methods for instruments to prevent further spread of vCJD and other TSEs (47)

  • Transmission of UK vCJD (BSE) and of New Guinea kuru (kCJD agent) to normal mice additionally emphasized the epidemic and a geographic-specific environmental sources of these agents, rather than some spontaneous conversion of host encoded PrP into an infectious (prion) form. (48,49). Removal of infected material from those environments has stopped these epidemics.

Transmission of HUMAN vCJD (left 3 pairs) & COW BSE (right 2 pairs) to normal mice both give identical strain-specific lesions (red). Arrows show strong hypothalamic involvement. Note this pattern is very different from sCJD & FU-CJD above. Markers for PrP, reactive glia (GFAP) and Keratan Sulfate (KS) on different pairs (in red).

Infection by vastly different TSE agents provoke the same PrP & PrP-res patterns.
  • Markedly different TSE agent-strains produce the same misfolded PrP-res bands if the host is kept constant. Something else, rather than PrP misfolded band patterns, must define the multiple agent strains.

This Western blot shows total cell PrP and PrP-res (+PK lanes) in mouse brains (left panel) with the different agents indicated. When transmitted to neural GT1 murine cultures (right panel), a different, cell specific pattern of PrP bands is seen. Despite this, TSE agents grown in GT1 cells for >1 year, when re-inoculated in mice, produce the original brain PrP pattern, i.e., the PrP patterns change in different environments, but the agent-strain is constant.

Transmission of many TSE strains to cultures with new agent assays

  • Development of a reliable, rapid tissue culture assay for many different CJD and scrapie strains. This has accelerated essential evaluations of infectivity, not just PrP-res. These assays have also facilitated analysis of many gradients and conditions not feasible in animal studies (48,49)

  • The doubling time of TSE infectious agents in cells is <1 day, but many days in mice (up to 33 days). Agent replication time is specific for, and dependent on, the infectious agent-strain. This rapid change in agent replication in GT1 cells demonstrates that TSE agents are released from inhibitory host immune controls; no model of PrP self-conversion can account for these precise agent replication features (52).

Characterization of TSE infectious particles

3D projection/rotations of TSE particle array

  • Gentle disaggregation of PrP (converting PrP-res back to sensitive PrP) does not alter infectivity o After PrP disaggregation, infectious particles band with a defined virus-like size and density. These particles also separate from the majority of host PrP (53-57).

  • Field flow fractionation and HPLC show infectious particles are ~25nm in diameter and ~5e6 daltons (58).

  • Infectious TSE particles, as many other viruses, resist nuclease digestion. As with viruses, disruption of nucleic acid-protein complexes (by GdnSCN or SDS) reduces TSE infectivity (47,53).

  • With disruption, long nucleic acids are released, including proteins that bind to and protect nucleic acid. These nucleic acids are in vast excess to those needed for a TSE genome; they include endogenous retroviral RNA and its protective gag protein (57-60).

  • More purified infectious material has 25nm dense particles ultrastructurally that do not bind PrP antibodies. Particles of 25nm also seen in viruslike arrays within some infected cells. These arrays are distinct and separated from intracellular PrP amyloid and normal membrane PrP (61).

  • Development of a rapid reproducible tissue culture assay for different CJD and scrapie strains. Our unique ability to titer various strains has accelerated essential evaluations of particle properties. These agent assays have facilitated analysis of many gradients and conditions not feasible in animal studies (50,62)

  • Discovery of new environmental circular DNAs of 1.8-2.4kb (Sphinx DNA elements) that concentrate in highly infectious preparations. These DNAs have been found in infectious fractions from cultures and brain and with a variety of CJD and scrapie agents (63). Parts of these sequences are linked to plasmids that infect gram-negative antibiotic resistant Acinetobacteria in the environment. Such sequences may have contribute to covert latent infection and neurodegeneration. All infectious preparations are contaminated with mitochondrial circular DNA as easily shown by PCR.

  • Most relevant is recent data showing that formation of cell-to-cell contacts recruits PrP (64). Furthermore, PrP functions as a receptor for TSE agents, and is part of an innate defense mechanism that can eliminate agent. PrP and PrP-res increase dramatically as the high titer FU-CJD agent is eliminated by 10,000 fold (4 logs) (65). This finding has great relevance for diverse ways misfolded proteins and amyloids can be educed, including various environmental and other stresses that contribute to, or bring out late onset neurodegenerative changes such as AD (66)

  • Even more remarkable is that destruction of all forms of detectable brain PrP does not result in any loss of infectivity (67). Thus PrP is very unlikely to be the causal TSE agent. The real infectious structure and its essential strain determining molecules remain an open and fundamental question.


Infectious TSE particles show a viral biology and structure that is inconsistent with prion protein models. Studies confined to PrP have limited discoveries of other proteins and nucleic acids that are probably structural elements of the infectious agent.

A protected and mutable nucleic acid genome is the most parsimonious way to explain all the distinct TSE agent strains, and its intact passage through the GI tract. In contrast, PrP is destroyed in the GI tract.


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