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VETM 422 Virology > Lecture 19: Prions > Flashcards

Lecture 19: Prions Flashcards

1
Q

Compare prions to viruses

A

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

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2
Q

What is the protein only hypothesis

A
  • 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
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3
Q

What does prion stand for

A
  • Proteinaceous infection particle = prion
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4
Q

What protein does PrPsc come from? What is its normal function

A
  • 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)

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5
Q

How does PrPsc replicate

A
  • PrP scrapies replicated by converting host protein into malformed/scrapie protein
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6
Q

Compare the structure of PrPc and PrPsc

A

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

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7
Q

How does PrPsc impact the immune system? How does that influence diagnostics?

A
  • No immune response – no Ig can be used for diagnosis
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8
Q

Features of prions/prion disease

A
  • 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
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9
Q

List 5 Transmissible Spongiform Encephalopathies

A

Transmissible Spongiform Encephalopathies
* BSE/Mad cow
* Scrapie
* Kuru
* Chronic wasting disease
* Creutzfeldt-Jakob disease

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10
Q

List 4 methods of diagnosing prion disease

A
  • Immunoblot
  • Enzyme linked immunosorbent assay
  • IHC/Histopath

not yet fully approved:
Real-time quaking induced conversion

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11
Q

What is the material and process of immunoblot diagnosis of prion disease

A
  • 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
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12
Q

What is the material and process of ELISA diagnosis of prion disease

A
  • 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)
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13
Q

What is the material and process of IHC/histopath diagnosis of prion disease

A
  • IHC/Histopath
    o Antibody based detection of PrPsc in tissue
    o Hematoxylin-eosin staining to see spongiosis
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14
Q

What is the material and process of Real-time quaking induced conversion diagnosis of prion disease

A
  • 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
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15
Q

What is the sample required for prion testing

A
  • Materials
    o Brain homogenates (confirm diagnosis post mortem)
     Use the obex region of the brainstem
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16
Q

Can an antemortem diagnosis be made for prion disease

A

o Ante-mortem diagnosis via
 Recto-anal mucosa associate lymphoid tissue biopsy
 Retropharyngeal lymph node biopsy
 Can diagnose chronic wasting disease or scrapie

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17
Q

What are the 3 etiologies of human prion disease

A
  • 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)
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18
Q

What animals are affected by scrapie

A

Target: sheep/goat/moufflon

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19
Q

What are the clinical signs of scrapie

A

Clinically: weeks to 6 months before death
* Mainly ataxia and pruritis
* Behavioural changes
* Incoordination
* Tremor

20
Q

How is scrapie transmitted

A

Shed: urine, saliva, feces, milk (long persistence in the environment – years)

Transmit: horizontally via contaminated pasture, vertically via transplacental

21
Q

What is the incubation time of scrapie

A

2-5yr

22
Q

Compare the CNS prion localization of atypical and classical scrapie

A

classic
Path: mainly cerebrum and cerebellum

atypical
Path: mainly in obex region and brainstem

23
Q

What animals are atypical scrapie affecting and what are the clinical signs?

A

Target: sheep, older animals
Clinically: ataxia and incoordination (pruritis uncommon)

24
Q

Why is atypical scrapie less concerning vs classical

A
  • Sporadic and non-contagious
25
Q

What is another name of Atypical Scrapie

A

Nor98

26
Q

Where is atypical scrapie located

A

Distribution: global, even in areas that don’t have classical scrapie

27
Q

How does diagnosis of atypical scrapie compare to classical

A

Diagnose: more bands on gel electrophoresis and of lower molecular weight

28
Q

How is classical scrapie controlled

A

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

29
Q

Explain the epidemiology of Classical BSE

A

Epi: Man made disease causing an epidemic in UK in 1990
* Feeding cow meat to cows
* Non contagious/no prion shedding

30
Q

What are the clinical signs of classical BSE

A

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

31
Q

What is the incubation time and animals mainly affected by BSE

A

Incubation: 2 – 8 years
Target: 4 – 5 year old dairy cow

32
Q

How is BSE controlled

A

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

33
Q

How is BSE transmitted

A

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

34
Q

Compare target age of sporadic and variant CJD in humans

A

Humans and Sporadic CJD
Target: 68 yo

Humans and varient CJD
Target: 28 yo

35
Q

Compare clinical signs of sporadic and variant CJD in humans

A

sporadic
Clinically: 6 months
* Dementia mainly

variant
Clinically: 14 months
* Psychiatric and ataxia

36
Q

Compare path of sporadic and variant CJD in humans

A

sporadic
Path: no florid plaques and only in CNS

variant
Pathology: florid plaques can be found in CNS and lymphoid
Transmit: blood transfusion

37
Q

What is the process of BSE surveillance

A

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

38
Q

How is atypical BSE transmitted, what animals does it target

A

Atypical BSE
Target: old >8yo cows, low frequency
Transmit: non infectious

39
Q

How is atypical BSE diagnosed

A

Diagnose: 2 forms of atypical: H and L type (based on molecular weight) – use western blot

40
Q

What animals does CWD target

A

Target: elk, mule deer, white tail deer, moose, reindeer = cervids (wild and farmed)
* Zoonosis is debated

41
Q

How is CWD transmitted

A

Transmit: horizontal and vertical via oral infection in soil

42
Q

How long is the incubation period of CWD

A

Incubation: 2 -4 year

43
Q

What are the clinical signs of CWD

A

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

44
Q

How is CWD shed

A

excretion in antler velvet
feces
recto-anal or mucosa-associated lymphoid tissue
urine

45
Q

What is the distribution of CWD

A
  • European and NA CWD not related
  • Increasing prevalence and geographic distribution– has been detected in camels

VETM 422 Virology (19 decks)

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