 Professor and Head of Neuropathology at Yale- Department of Surgery and Faculty of Neurosciences and Virology  Focuses on dementia with emphasis on TSEs  “We think it is most likely the host PrP is a required receptor for TSE viruses, and that viral PrP- membrane interactions ultimately cause a pathological PrP response. Ongoing experiments are designed to test this viral hypothesis”

Kohtaro Miyazawa, Kaitlin Emmerling, and Laura Manuelidis Journal of Cellular Biochemistry 112: 3630–3637 (2011) “Substantial and often ignored evidence indicates that PrP-res is part of a pathological response to infection, rather than the causal agent that incites disease. Many viruses require a specific host protein for entry and replication, and infectious TSE particles similarly require host PrP for effective spread from ce ll to cell. Merely increasing PrP, even from a different species, enhances transmission of foreign TSE. Diverse published data that are inconsistent with an infectious form of PrP “

(i)the variety of unique and mutable agent strains, a property of nucleic acid not protein (ii)TSE agents breed true in various tissues, cell cultures, and cross-species transmissions whereas PrP patterns do not (iii) a preventable environmental source of infection in which epidemic outbreaks disappear after infectious material is removed (as in BSE and kuru) strongly implicates a foreign source of infection rather than a ‘‘spontaneously generated’’ host PrP self-conversion into an infectious form (iv) infection elicits early innate immune responses that indicate host recognition of an invading foreign entity and these responses, not educed by host PrP, occur well before PrP-res begins to accumulate and can be protective

(v) microglia with barely detectable PrP, and no PrP-res, contain high levels infectivity (vi)all detectable forms of PrP are digested in the gastro intestinal tract yet the invasive infectious particle (as many conventional viruses) is not destroyed. (vii) infectious particles of viral size ( 25nm diameter) with protected nucleic acids can be separated from the majority of host PrP and other proteins, and GdnSCN disruption of these particles into protein and nucleic acid components reduces infectivity by>99.8%. Some of these released nucleic acids have been sequenced such as capsid-protected endogenous retroviral RNAs, mitochondrial DNA, and newly discovered circular Sphinx DNAs of1.8kb with large sequence regions that are not in the database (viii) High CJD infectivity remains after the prion protein is destroyed with a protease.

 Prion strain- defined as “infectious isolates that, when transmitted to identical hosts, exhibit distinct prion-disease phenotypes”  Prion strain diversity first seen in goats  PrP-c and PrP-sc can be unglycosylated, monoglycosylated or diglycosylated. Aguzzi, A., Heikenwalder, M., Polymenidou, M. (2007). Insights into prion strains and neurotoxicity. Nature. 8:

Aguzzi et al. (2007). Insights into prion strains and neurotoxicity. Nature.8):

 Manuelidis et al (1997) ◦ Several strains, same PrP amino acid sequence ◦ Different phenotypes  Due to PrP protein or hidden virus?  Experiment ◦ Succeeded in changing a CJD strain into a strain that produced plaques and cerebellar lesions  Evaluated host recognition by responses of microglia and astrocytes  Used inbred mice, guinea pigs, and rats. Manueldis, L., Fritch, W., Xi, Y. (1997). Evolution of a strain of CJD that Induces BSE-Like Plaques. Science. 277(5322):

 Authors state “Species barrier and host responses (reactive microglia, astrocytes, and development of new strain that can produce plaques/lesions from a strain that couldn’t before) to the foreign agent are too complex to just be explained by the host’s PrP sequence”

 “TSE strains maintain their identity despite various changes in prion protein. This fact strongly implicates a relatively stable but mutable viral genome” (from Yale bio page)  Wanted to see whether PrP-res itself encoded intrinsic infectivity characteristics  Slow SY strain and fast virulent FU CJD strain infect GT1 cells into various cell lines ◦ Kept phenotypes through passage but remained indistinguishable by PrP-res banding or glycosylation patterns  FU had different PrP-res patterns in different cell lines  Still had same incubation time and clinical features ◦ Amount of PrP-res was not quantitatively related to infectivity  “It is the biology of these agents: their evolution spread, cell specificity, latency, virus-like interference capabilities and occusional mutation which continues to indicate a viral causative agent” ◦ Arjona et al. (2004). Two Creutzfeldt-Jakob disease agents reproduce prion protein-independent identities in cell cultures. PNAS. 101(23):

 Authors argue “These findings (two strains that have different phenotypes but same PrP- res banding or glycosylation patterns) are problematic for the prion hypothesis where abnormal PrP folding or glycosylation, and hence PrP-res band patterns, are postulated to encode each agent strain”

◦ PrP(Sc) prion populations with distinct phenotypes but same amino acid sequence..are distinct strains. ◦ Strain identity encoded by conformation of PrP(Sc) ◦ Brain-derived 22L prions are able to infect R33 cells (R33 competent) AND PK1 cells in the presence of the swainsonine (swa resistant). ◦ 22L prions retained these characteristic s including swa resistance when transferred from brain to R33 cells. ◦ However, when transferred from the R33 cells to PK1 cells, they gradually became R33 incompetent and swa sensitive, unless the transfer was in the presence of swa, in which case swa resistance and R33 competence were retained. Mahal et at. (2010). Transfer of a prion strain to different hosts leads to emergence of strain variants. PNAS. 107(52):

 PrP(Sc) with swa-resistant/R33-competent and PrP(Sc) swa- sensitive/R33-incompetent prions had different conformational stabilities.  When R33-incompetent/swa-sensitive prions were propagated in brain, their properties gradually reverted to those of the original brain-derived 22L prions.  Conclusion: 22L prion populations are heterogeneous and that distinct prion variants are selected in different cellular environments. ◦ ….viral hypothesis would say….??? Mahal et at. (2010). Transfer of a prion strain to different hosts leads to emergence of strain variants. PNAS. 107(52):

 Showed prions can adapt to survive in a new host environment ◦ When transferring from one cell line to another, prion properties chance  “Darwinian Evolution without DNA” ◦ Prions can develop mutations  drug resistance ◦ Fold in different ways  new strains ◦ Transferred to a new host  which strain ‘wins’? ◦ …..same data different interpretations??

◦ Study by Cancellotti et al., (2013) showed that the passage through mice that expressed a PrP that either partially or completely lacked N-glycan affected the phenotypic characteristics of at least one TSE strain ◦ Quasi species hypothesis  Several PrP-sc conformations in infectious innoculum. One best suited for new host is selected for  Problem- there isn’t a lot of evidence that a large number of conformations exists in an innoculum Poggiolini, I., Saverloni, D., Parchi, P. (2013). Prion Protein Misfolding, Strains, and Neurotoxicity: An Update from Studies on Mammalian Prions. International Journal of Cellular Biology Soto, C and Castilla, J. (2004). The controversial protein-only hypothesis of prion propagation. Nature medicine

 PrP expressed and purified from E. coli.  …refolded and kept in solution using 1%SDS  …spontaneously forms fibrils when SDs is diluted to 0.02%  …seeding with different brain extracts results in faster fibrillation  …go to paper

 Evidence of viral presence  Endogenous Viral Complexes with long RNA cosediment with the infectious agent of Ceutzfeldt-Jacob Disease

 Strain variation, exponential replication, tissue specificity, latency, resistance to treatment, and non-inflammatory response…all characteristics of retroviruses  LTRs present in infectious samples  Long terminal repeats (LTRs) are identical sequences of DNA that repeat hundreds or thousands of times found at either end of retrotransposons or proviral DNA formed by reverse transcription of retroviral RNA. They are used by viruses to insert their genetic material into the host genomes.  Endogenous retroviral intracisternal A particle (IAP) genome was identified

 IAP are a class of retrovirus  Founf in infectious sample but also in non-infectious sample  IAP are highly resistant to forms of treatment like SDS and chaotropic salts  High resistance is due to IAP gag proteins  gag protein found indicating a virus particle, not just random IAP RNA  Sensitive to guanidinium chloride treatment

◦ Characteristic of the viral particles  Core-like viral density of g/cc  Viral size of 120S and a diameter of ~30nm  >99% of starting prion protein can be separated from the viral agent and still have infectivity

 Absence of association between the incidence of BK virus and sporadic Creutzfeldt-Jakob disease. Jeong BH1, Lee JH, Cho HJ, Kim YS. Intervirology. 2013;56(3):  OBJECTIVE:  To determine the relationship between BK polyomavirus (BKV) and sporadic CJD.  MATERIALS AND METHODS:  We investigated the prevalence of BKV in urine samples from 94 sporadic CJD patients and 54 other neurological disease (OND) patients using polymerase chain reaction.

 BK virus:  -member of the polyomavirus family  -first isolated in 1971  -generally asymptomatic except when immunosuppressed  - carried by an estimated 80% of the population  - no treatment except to switch immunosuppression drug  - transmission possibly through saliva and urine

 RESULTS:  BKV DNA was detected in 16 (17%) and 9 (16.7%) urine samples from sporadic CJD and OND patients, respectively. There was no significant difference in the incidence of BKV infection between Korean sporadic CJD and OND patients (p = ). In order to investigate the genotypes of BKV, we analyzed 22 BKV isolates obtained from Korean patients by DNA sequencing and nucleotide sequence analysis. Three distinct subtypes, namely I, III, and IV, were found in 66.7, 22.2, and 11.1% of 9 BKV isolates from OND patients, whereas subtypes I and IV were detected in 76.9 and 23.1% of 13 BKV isolates from sporadic CJD patients. Interestingly, subtype III was not detected in sporadic CJD patients. Significant differences in the frequency of BKV genotypes were not observed between sporadic CJD and OND patients.  CONCLUSIONS:  These results indicate that BKV may not play an important role in the pathogenesis of prion diseases.

 Digestion of PrP in sample with protease K does not reduce infectivity  Manuelidis 2014

 CJD killed our son…