Summary of Virological Methods

1. 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)

2. 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

3. 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

4. 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

5. 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

6. 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

7. Nucleic acid is not necessary for infection

8. Stabilities of the scrapie agent and viriods (PSTV): * Chemical Treatment: Concentration PSTV Scrapie Et2PC 10-20mM (-) + NH2OH 0.1-0.5mM + - Psoralen 10-500µg/ml + - Phenol Saturated - + SDS 1-10% - + Zn2+ 2mM + - Urea 3-8M - + Alkali pH 10 (-) + KSCN 1M - + Enzymatic Treatment: Concentration PSTV Scrapie RNAse A 0.1-100µg/ml + - DNAse 100µg/ml - - Proteinase K 100µg/ml - + Trypsin 100µg/ml - + + = inactivated; - = no change in infectivity

9. 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

10. 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

11. 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

12. 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

13. Spongiform appearance Amyloid deposition in brain

14. 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

16. 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

17. 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

18. 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

19. 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 raisedthe specter of a potentially large population being at riskto develop this prionosis. None of the prionoses currently havean effective treatment. Recently, vaccination has been shownto be effective in mouse models of another neurodegenerativecondition, namely Alzheimer’s disease. Here we reportthat vaccination with recombinant mouse prion protein delaysthe onset of prion disease in mice. Vaccination was performedboth before peripheral prion exposure and after exposure. Adelay in disease onset was seen in both groups, but was moreprolonged in animals immunized before exposure. The increasein the incubation period closely correlated with the anti-prionprotein antibody titer. This promising finding suggests thata similar approach may work in humans or other mammalian speciesat risk for prion disease. Am. J. Pathol. 2002, 161:13-17

20. 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

21. 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