Lecture 19: Prions Flashcards
(45 cards)
Compare prions to viruses
Prion
No genetic info = NO PCR
Protein only
Bases that denature proteins = efficient
- Urea
- Guanidinium salt
- NaOH
No adaptive immune response
- No Ig or T cell activation
Virus
Genetic info = PCR
DNA/RNA/Protein/Lipid coat
Inactivate with
- UV
- Formaldehyde
- Alcohol
- Autoclave at 121C
Immune response include
- Inflammation
- Ig
What is the protein only hypothesis
- Prions self-replicated without nucleic acids by converting a normal protein to misfold (converting to protease resistant protein)
- Proportional increase of infectivity with amount of ‘prion’ protein dose = infectious disease
What does prion stand for
- Proteinaceous infection particle = prion
What protein does PrPsc come from? What is its normal function
- Cellular prion protein = all mammals express
membrane glycoprotein
o Normally produced, mainly in brain
o Can change conformation to PrPsc
o Normal function = synaptic transmission, circadian rhythm, copper transport and release, signalling, neuroprotective
Functions are redundant – if it is knocked out = not significant
* Prion protein scrapie = infectious form (PrPsc)
How does PrPsc replicate
- PrP scrapies replicated by converting host protein into malformed/scrapie protein
Compare the structure of PrPc and PrPsc
PrPc
o Structure: alpha helix, soluble, proteinase K sensitive, non infectious
PrPsc
o Structure: beta sheets (that can aggregate and form fibrils), insoluble, partially proteinase K resistant (the core of the fibril is resistant), infectious
o Same primary structure/amino acid sequence as PrPc
How does PrPsc impact the immune system? How does that influence diagnostics?
- No immune response – no Ig can be used for diagnosis
Features of prions/prion disease
- No nucleic acids (resistant to UV light degradation)
- Resistant to proteases
- No immune response – no Ig can be used for diagnosis
- Fatal
- Transmissible
- Cause spongiform neurodegeneration
- Long incubation (years) and a short clinical phase (death in months)
- No treatment or prophylaxis
List 5 Transmissible Spongiform Encephalopathies
Transmissible Spongiform Encephalopathies
* BSE/Mad cow
* Scrapie
* Kuru
* Chronic wasting disease
* Creutzfeldt-Jakob disease
List 4 methods of diagnosing prion disease
- Immunoblot
- Enzyme linked immunosorbent assay
- IHC/Histopath
not yet fully approved:
Real-time quaking induced conversion
What is the material and process of immunoblot diagnosis of prion disease
- Immunoblot
o Materials: proteinase K digested tissue homogenate
o Method: separate protein according to molecular weight in a gel matrix
Electro transfer to a membrane and incubate with an Ig
Identify ig reaction
What is the material and process of ELISA diagnosis of prion disease
- Enzyme linked immunosorbent assay
o Material
o Method: digest homogenate with proteinase K
Ig that recognizes PrPc in the 96 well plate
Add another Ig that binds the first Ig
‘Sandwich ELISA’
Only colour reaction if substance binds the first antibody
Only PrPsc is bound by the first ig (because PrPc is degraded by proteinase k)
What is the material and process of IHC/histopath diagnosis of prion disease
- IHC/Histopath
o Antibody based detection of PrPsc in tissue
o Hematoxylin-eosin staining to see spongiosis
What is the material and process of Real-time quaking induced conversion diagnosis of prion disease
- Real-time quaking induced conversion
o Identify in vitro conversion
o Similar to qPCR for proteins
o Use normal form of prion protein as substrate
o Mix with serial dilution of potentially infected brain homogenate
o Add fluorescent dye that binds the PrP scrapie fibrils
o Repeat cycles of shaking and incubation
o Initial ‘seed’ will converted the substrate aggregated form
o Detect/measure fluorescence
What is the sample required for prion testing
- Materials
o Brain homogenates (confirm diagnosis post mortem)
Use the obex region of the brainstem
Can an antemortem diagnosis be made for prion disease
o Ante-mortem diagnosis via
Recto-anal mucosa associate lymphoid tissue biopsy
Retropharyngeal lymph node biopsy
Can diagnose chronic wasting disease or scrapie
What are the 3 etiologies of human prion disease
- Sporadic: 85-90% of cases, spontaneous conversion
- Inherited: germline mutations – the only type that can be diagnosed before clinical signs (tests as indicated by family history)
o Familial CJD
o Gerstmann-Straussler-Scheiniker Syndrome
o Fatal Familial insomnia - Acquired: contaminated tissues/ingestion of contaminated food
o Iatrogenic/kuru/variant CJD (from BSE)
What animals are affected by scrapie
Target: sheep/goat/moufflon
What are the clinical signs of scrapie
Clinically: weeks to 6 months before death
* Mainly ataxia and pruritis
* Behavioural changes
* Incoordination
* Tremor
How is scrapie transmitted
Shed: urine, saliva, feces, milk (long persistence in the environment – years)
Transmit: horizontally via contaminated pasture, vertically via transplacental
What is the incubation time of scrapie
2-5yr
Compare the CNS prion localization of atypical and classical scrapie
classic
Path: mainly cerebrum and cerebellum
atypical
Path: mainly in obex region and brainstem
What animals are atypical scrapie affecting and what are the clinical signs?
Target: sheep, older animals
Clinically: ataxia and incoordination (pruritis uncommon)
Why is atypical scrapie less concerning vs classical
- Sporadic and non-contagious
