Transcript Sub-viral Agents

Summary of Virological Methods
• Virus tittering methods (infectivity)
– Plaque assay
– Tissue culture infectious dose (TCID50)
• Hemagglutination (HA): hemagglutinin
• Immunofluorescence staining(Diagnosis)
• Serology methods (diagnosis)
– Enzyme-linked immunosorbent assay (ELISA)
– Immunoblot assay
• Molecular methods
– Polymerase chain reaction (PCR)
– Reverse-transcription polemerase chain reaction (RT-PCR)
– Real-time PCR (quantitative PCR)
Sub-viral Agents
• Satellites: small ss RNA, 500-2000 nucleotides, long
circular
-- Satellite virus: encodes its own protein coat
-- Satellite RNAs (Virusoids):use coat protein of the helper
virus
• Viroids: a single molecule of RNA, 246-357 nucleotides.
common plant pathogens (20-25 identified)
• Prions: small proteanous infectious particles
Satellites
• Depend on another virus for
replication (helper virus)
• Replicate in cytoplasm by RNA
dependent RNA polymerase
(common in plants)
• No similarity to the helper virus
in genome (unlike Defective
interfering particle)
• Interfere with the replication of
helper virus
• Cause distinct disease alone in
plants
Viroids
• Encodes no proteins
• Replicate by host cellular RNA
polymerase II (Plant nucleolus)
• No coat (capsid or envelope)
• No proteins made
• No helper virus needed
• Rod-like, high secondary
structure
• Conserved region and a
ribozyme region (catalytic RNA
molecule and cleave RNA)
• Potato spindle tuber viroid and
cadang-cadang coconut viroid
Hepatitis delta virus (HDV)
• Infects human
• ss RNA, 1700 nucleotides,
circular
• Combined features of satellites
and viroids
• Encodes delta antigen
• RNA and delta antigen are
packaged in hepatitis B virus
(HBV) particles
• Has ribozyme activity
• Potentiate the pathogenic
effects of HBV
• HBV vaccination can prevent
the infection
• Common in indigenous natives
of South America
Prions
• Only protein, no nucleic
acid
• Sensitive to proteases
• Resistant to nuclease and
procedures modify nucleic
acids
• Modified form of a normal
cellular protein
• Encodes by a single exon
of a single copy gene
Nucleic acid is not necessary for infection
Stabilities of the scrapie agent and viriods (PSTV):
*
Chemical Treatment:
Et2PC
NH2OH
Psoralen
Phenol
SDS
Zn2+
Urea
Alkali
KSCN
Enzymatic Treatment:
RNAse A
DNAse
Proteinase K
Trypsin
Concentration
10-20mM
0.1-0.5mM
10-500µg/ml
Saturated
1-10%
2mM
3-8M
pH 10
1M
Concentration
0.1-100µg/ml
100µg/ml
100µg/ml
100µg/ml
+ = inactivated; - = no change in infectivity
PSTV
(-)
+
+
+
(-)
PSTV
+
-
Scrapie
+
+
+
+
+
+
Scrapie
+
+
Diseases Associated With Prions
• Scrapie: slow, fatal disease of sheep and goats, transmissible by
close contact with infected animals and pasture (placental tissue)
or direct sheep-to-lamb transmission, known for several hundreds
of years
• Creutzfeldt-Jacob disease (CJD): described in 1920, Human,
occurs sporadically, transmissible to chimpanzees and other
monkeys by inoculation into brain
• Kuru, first described in 1950s, human in New Guinea Fore Tribe,
only in female and adolescents, associated with ritual cannibalism
• Bovine spongiform encephalitis (BSE) (1986), transmissible
mink encephalopathy (TME) and chronic wasting disease (CWD)
of deer and elk
• New variant-CJD:described in 1996, most likely linked to
exposure to BSE
PrPsc is associated with scrapie infectivity
• Purification of scrapie infectivity results in
preparations highly riched for PrPsc
• Purification of PrPsc results in enrichment
of scrapie activity
• Purification of PrPsc by SDS-PAGE also
recovers infectivity
• PrPsc can be denatured and renatured
without loss of infectivity
How can a protein be infectious
• Normal protein PrPc(cellular ):
alpha helical, no beat sheet
• Predominantly on the surface of
neurons, synaptic function
• Scrapie protein PrPsc: dominantly
beat sheet
• PrPsc is transdominant which
convert PrPc to PrPsc in an
exponential fashion
• When PrPc is incubated with
PrPsc, the conformation of PrPc is
changed to PrPsc
• Mice not expressing PrPc are not
susceptible to prion disease
Characteristics of diseases caused by prions
• Long incubation period, slow process
• Loss of motor control, dementia, paralysis wasting
and eventual death
• Neuropathology: neuronal death, spongiform
changes (holes in brain tissue), glial and astrocyte
proliferation, no inflammatory response
• Amyloid plaque in brain tissue: insoluble protein
aggregates
• Both infectious and hereditary, and also sporadic
Spongiform appearance
Amyloid deposition in brain
Pathogenesis
• Enter through gut wall (peyers patches): mucosal lymphoid
tissue
• Take up by lymphoid cells and travel to other lymphoid tissue
• Travel to the nerve and propagate, back up to axon, spinal
cord and eventually brain
• SCID mice are resistant to prion challenge, confirming the
importance of the lymphoid system in the pathogenesis
• Susceptibility depend on both prion inoculum and the normal
prion protein gene
• Transmission across species occur after very long incubation
time
• Mutated PrPc gene more susceptible to the disease
BSE (mad cow disease)
• In 1986 UK
• Meat product and bone
meal (sheep)
• Massively expensive
culling program
• 10% of calves born to the
cows die of BSE will die
of BSE themselves
• Environmental
contamination, hard to
eradicate
• No BSE in the US, but
TME described
Incidence of vCJD in human
• BSE transmissible to many species
experimentally (sheep, pigs,
macaque monkeys, cats) by eating
tainted beef
• In 1996 a new variant CJD
recognized in UK
• Strikingly early age (14 yrs and
up)
• As of June 2001, 100 confirmed
cases
• Likely cause is BSE (by strain
typing of v-CJD
• Bruce: Nature paper, linked BSE
to human vCJD
• Oral ingestion, but why incidence
is still very low
Treatment
• Reduce the expression of normal PrPc
• Stabilze the PrPc conformation
• Destablize the PrPsc conformation
~ Several vaccines to Alzheimers (amyloid
plaques) are in clinical trials
• Degradation of proteins
Vaccines?
Immunization Delays the Onset of Prion Disease in Mice
Einar M. Sigurdsson*, David R. Brown, Maki Daniels, Richard J. Kascsak, Regina Kascsak,
Richard Carp, Harry C. Meeker, Blas Frangione* and Thomas Wisniewski*¶
From the Departments of Psychiatry,*Pathology,and Neurology,¶New York University School of Medicine, New York, New York;
the New York State Institute for Basic Research in Developmental Disabilities,Staten Island, New York; and the Departments of
Biology and Biochemistry,University of Bath, Bath, United Kingdom
Abstract
The outbreak of new variant Creutzfeldt-Jakob disease has raised the specter of a potentially large
population being at risk to develop this prionosis. None of the prionoses currently have an effective
treatment. Recently, vaccination has been shown to be effective in mouse models of another
neurodegenerative condition, namely Alzheimer’s disease. Here we report that vaccination with
recombinant mouse prion protein delays the onset of prion disease in mice. Vaccination was performed
both before peripheral prion exposure and after exposure. A delay in disease onset was seen in both
groups, but was more prolonged in animals immunized before exposure. The increase in the incubation
period closely correlated with the anti-prion protein antibody titer. This promising finding suggests that a
similar approach may work in humans or other mammalian species at risk for prion disease.
Am. J. Pathol. 2002, 161:13-17
Evidence to support protein mode of infection
• Unusual small target size for ultraviolet and
ionizing radiation
• Low ratio of nucleic acids to infectious material
• Resistance of infectivity to agents which modify or
damage nucleic acids but infectivity is susceptible
to reagent which destroy proteins
• Failure to identify a specific nucleic acid either in
infected brain or in prion preparations using a
variety of sophisticated techniques
Replication
• Transcription of the short-form
of HDAg
• HDAg is required for RNA
replication
• Antigenomic RNA undergo
autocleavage and to yield
circular by a host enzyme, which
used as a template for genome
replication
• Transcription of long-form
HDAg, inhibits RNA replication
• Host enzymes involved in the
interaction with envelope protein
and virion formation