Lecture 19: Prions Flashcards
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
What is another name of Atypical Scrapie
Nor98
Where is atypical scrapie located
Distribution: global, even in areas that don’t have classical scrapie
How does diagnosis of atypical scrapie compare to classical
Diagnose: more bands on gel electrophoresis and of lower molecular weight
How is classical scrapie controlled
Control of Classical scrapie
* Many genotype of prion proteins due to SNPs
* Some genotypes are resistant (some are very susceptible)
* Can breed sheep with highly resistant genotype
* But even if resistant to classical scrapie they can still get atypical scrapie
Explain the epidemiology of Classical BSE
Epi: Man made disease causing an epidemic in UK in 1990
* Feeding cow meat to cows
* Non contagious/no prion shedding
What are the clinical signs of classical BSE
Clinically
* Behaviour change: nervous, teeth grinding, frequent nose licking, tremor
* Sensory system abnormality: hypersensitive to touch, light, noise
* Locomotion abnormalities: ataxia (hindlimb), pacing, hypermetria, can’t get up
What is the incubation time and animals mainly affected by BSE
Incubation: 2 – 8 years
Target: 4 – 5 year old dairy cow
How is BSE controlled
Control: ban feeding of ruminants to ruminants, also ban feeding specified risk material (CNS/dorsal and trigeminal ganglia/eye/tonsil/distal ileum of all ages) from cows to humans or animals
Geography
* Last case in CA 2015
How is BSE transmitted
Transmit: all only via eating contaminated meat (not shedding)
* Feeding cattle with atypical scrapies can cause
* Eating contaminated ruminant meat can cause infection to pigs, people, cats, exotic ungulates
* Dogs resistant to BSE infection
Compare target age of sporadic and variant CJD in humans
Humans and Sporadic CJD
Target: 68 yo
Humans and varient CJD
Target: 28 yo
Compare clinical signs of sporadic and variant CJD in humans
sporadic
Clinically: 6 months
* Dementia mainly
variant
Clinically: 14 months
* Psychiatric and ataxia
Compare path of sporadic and variant CJD in humans
sporadic
Path: no florid plaques and only in CNS
variant
Pathology: florid plaques can be found in CNS and lymphoid
Transmit: blood transfusion
What is the process of BSE surveillance
Surveillance
1. Screen with western blot or ELISA – test any animals with clinical signs (>30mo, dead, down, dying, diseased)
2. Non-negatives will be sent to CFIA in Lethbridge + sasme screening will be used (western blot)
3. All positives = ‘suspect for BSE’
4. CFIA confirmatory testing via IHC, OIE, Western blot
5. Animals that are born 12 months before or after + case of BSE/exposed to saem feed as BSE animals for first year of life = cull and test
How is atypical BSE transmitted, what animals does it target
Atypical BSE
Target: old >8yo cows, low frequency
Transmit: non infectious
How is atypical BSE diagnosed
Diagnose: 2 forms of atypical: H and L type (based on molecular weight) – use western blot
What animals does CWD target
Target: elk, mule deer, white tail deer, moose, reindeer = cervids (wild and farmed)
* Zoonosis is debated
How is CWD transmitted
Transmit: horizontal and vertical via oral infection in soil
How long is the incubation period of CWD
Incubation: 2 -4 year
What are the clinical signs of CWD
Clinically: 4 month – 1 year
* Progressive weight loss
* Behavioural change (no fear of humans)
* Hypersalivation, pneumonia
* Depression, teeth grinding
* Ataxia, difficulty swallowing
* Polydipsia and polyuria
How is CWD shed
excretion in antler velvet
feces
recto-anal or mucosa-associated lymphoid tissue
urine
What is the distribution of CWD
- European and NA CWD not related
- Increasing prevalence and geographic distribution– has been detected in camels
