Prions

Theory, detection, control

Sheep and goat prion disease

scrapie

2-5yrs

Cow prion

BSE

Bovine Spongiform encephalopathy

(mad cow)

zoonotic → vCJD = varient of Creutzfeldt-Jakob Disease

4-5yrs

Cervid prion

chronic wasting disease

2-4 ys

TSEs

transmissible spongiform encephalopathies

• not a disease of young animals due to long incubation period

Prions - hard to destroy

differentiates them from conventional bacteria, viruses and parasites

• host protein predominanty found in the brain

• Prp^c → Prp^Sc } Scrapies

• Prp^Sc = disease-associated isoform

• alpha helix chain → beta pleated sheat

  • Beta-pleated sheets → difference in seconadary and tertiary struc

• misfolding disease

Heat, pressure or ionising radiation (breaks DNA and RNA) do not affect infectivity/break down prions

Prion replication

1. Initial PrPSc seed introduction (post translational modification)

2. PrPSc acts as template to convert PrPC into PrPSc → “self replication”

3. PrPSc increases over time → fibrils and plaques

• difficult to degrade (increase protease resistance)

• aggregates → neurotoxic

• longer half life

Testing PrPC protein theory

PrPC knockout mite injected with brain tissue → no PrPC to convert → PrPSc is broken down → negligible neuronal damage

PrPSc biochemical properties [2]

1. beta pleated sheets prominent in 2ndary structure

   • PrPC alpha helices

2. Aggregates → Proteolytic resistance

PrPSc biological properties [2]

1. PrPsc neurotoxicity

2. Self-replicating (acts as template)

Prion clinical signs

• Altered behaviour and motor function

  • Neurodegeneration in specific brain parts and synapse damage

  • Do not respond normally to pain

• Spongiform nature → NOT due to loss of neurons

  • neuron vacuolation due to prion induced change in neuron metabolism

  • Vaculation is reversible if cured in experimental models

Diagnosing prion disease

• No pathognomic clinical signs

• Brain vacuolation not pathognomic

• No antibodies → self reactive T cells depleted before birth

  • No T cell help → no B cells → no IgG

• Self → no DNA or RNA probes

• PrPSc is surrogate marker → use biochemical differences to distinguish from PrPC

• PrPSc aggregates → generate antibodies to antibodies to human PrPSc experimentally and inject in lab animal (foreign protein)

  • Visualise aggregates experimentally

3 diferent forms of PrPC- post-translational modification

• Prion protein has 2 sites of glycosylation

  • attachment can be lipids, glycans, glycolipids onto protein backbone

• Human antibody binds to prion protein

• 3 bands on gel to correspond to each type- Western blot (electrophoresis + antibody probe)

Lightest (travels the most) → heaviest (travels the least)

1. Non glycosylated PrPC

2. 1 glycosylated site

3. 2 glycosylated site

ALL can be converted into PrPSc

Separating PrPSc from PrPC

• Split sample into 2

• Use protease digestion on one sample

• use the other as a positive control

• Prion protein will be protease resistant and so there will be lines of protein on Western blot

  • 3 lines form for each variant (glycosylation)

• If only PrPC → ALL will be digested

  • NO LINES FORM

Route of transmission of prions

Oral

• GIT → bloodstream

• PrPSc accumulates and replicates in peripheral immune system

  • liver

  • spleen

  • GALTs (gut associated lymphoid tissues)

• PrPSc thought to invade periperal neurons

  • retrograde transmission to the spinal cord → brain

  • long travel → long incubation

vCJD

• remove from food chain

• prevent blood transfusions

  • test blood

• sterilise brain surgical equipments

Scrapie control problems

• remains in soil in infected pasture → animals eat infected soil

• no vaccine or effective treatment

  • resistant to commercial disinfectants

• cull infected animals

• NOTIFIABLE → movement restrictions on flock (2 years)

  • if no new cases then quarantine ends

  • restriction period extended from date of most positive result

• farmers reluctant to have animals tested

scrapie control → national breeding plans

• selective breeding of scrapie resistant sheeps

• mutations at codons 136 154 and 171

• different levels of genetic resistance

• not popular with farmers

• For BSE → also transgenic cattle with PrP gene knocked out

BSE transmission

• causative agent → infected meat and bonemeal

• high protein supplement → increased milk rates and growth yields

• completely different to scrapie and vCJD → no environmental factor

• sporadic disease in cow

• control → ban meat and bonemeal (MBM)

  • only infected MBM can cause infections

how kill prion

121C 1 hr

scrapie prions differ from BSE/CWD/CJD prions → how test?

inject brain tissue from different prion diseases into mice:

• different but highly reproducible patterns of brain attack

• different incubation period (shortest to longest):

  • BSE → CWD → scrapie [survival time in days]

  • similar kill curve of BSE, CJD and FSE (feline SE)

    • FSE, CJD come from BSE and so are similar

• dogs seem to be resistant to prions