Viral Detection

Viral DetectionBy: Douglas Tran

Past research States Prion hypothesis and past research of

no past viral genetic material detected States the fact that mitochondrial DNA has

been found in subsequent studies States that long RNAs cores were also

detected Very similar to retroviral cores

Endogenous Viral Complexes with long RNA cosediment with the agent of Ceutzfeldt-Jacob Disease

CJD Retroviral Theory Strain variation, exponential replication, tissue

specificity, latency, resistance to treatment, and non-inflammatory response

LTRs detection experience found LTRs present in infectious samples

Endogenous retroviral intracisternal A particle (IAP) genome were identified by using cDNAs that were created from the LTRs of infectious factions

Endogenous Viral Complexes with long RNA cosediment with the agent of Ceutzfeldt-

Jacob Disease

IAP RNA cosediement found (The RNA that is being detected) IAP are a class of retrovirus In Infectious sample In non-infectious sample

IAP are highly resistant to forms of treatment like SDS and chaotropic salts High resistance is due to IAP gag proteins Isolation of this gag protein would help solidify the

idea of virus particle, not just random IAP RNA


Jacob Disease

Source of materials and purification of CJD infectivity Syrian hamster IAP genome Polyclonal antibodies that bind the IAP gag protein 12 CJD infected hamster brains

Purification: samples made devoid of PrP-res Samples also treated with nuclease (allows for isolation of

nuclease resistant genetic material) Extraction of RNA

Elimination of DNA in sample via various methods (DNAase)


Jacob Disease

RNA/PCR amplification of IAP sequences Usage of various IAP primers for the production of

subsequent cDNA syntheses IAP RNA from cells -> cDNA-> DNA amplification via IAP

primer Probe hybridization to Southern Blots

DNA probes Generated from recombinant clone of Syrian Hamster IAP

Western blot Polyclonal rabbit anti-mouse IAP gag antibodies


Jacob Disease


Jacob Disease


Jacob Disease

• Discussion: • Was able to isolate both IAP RNA and

RNA gag in infectious samples • Both are resistance to degradation of

nuclease, which shows characteristic of retroviral

• Results refuted the assumption of prion infection being devoid of nucleic acid• ~6kb IAP sequences

• No clear evidence of a CJD specific sequence of IAP RNA• If IAPs are involved with the CJD

nucleic acid, it is either co-packed in the core or uses IAP products to proliferate

• Second theory: Completely independent CJD viral complex, only similar to IAP • Supported by presence of gag-like

proteins

States the assumption of virus infectivity theory Characteristic of the CJD infectious agent

Core-like viral density of 1.27-1.28 g/cc Viral size of 120S and a diameter of ~30nm >99% of starting prion protein can be separated from the viral

agent Primary investigation: Separation and isolation of the

primary components of the viral protein (testing different methods) PrP Nucleic acid-binding proteins Gag protein

Viral Particles are required for infection in neurodegenerative Creuzfeldt-Jakob disease

Method Centrifuge isolation of supernatant and pellets

after elimination of the majority of PrP Immunoblots (Western blot method used for

protein isolation) Polyclonal antibodies


Results show that a specific nucleic acid and one or more binding proteins are intrinsic components of the CJD virus Independently have low infectivity, but together form a

complex that is the main infective agent Infectivity is not Prp dependent,

SDS treatment eliminates the majority of Prp, but infectivity is still high

Gdn-HCI treatment who greatly lower infectivity due to elimination of the viral components There is still retention of PrP in Gdn-HCI samples


Begins paper by stating examples of virus that have similar and/or the same characteristics of CJD viral infection

States theory involving PrP as the viral receptor and that after interaction with virus it causes the formation of PrP-res

Lists the common flaws in Prion Hypothesis

Nuclease resistant circular DNAs copurify with infectivity in scrapie and CJD

Primary goal: Isolation and identification of circular ssDNA

via generalized detection method Ф29 polymerase to enhance the replication of

the circular ssDNA Circular DNA hypothesis is based off CJD’s

similarity with Torgue tenovirus, which has circular DNA It is also based on Circular DNA’s ability to cause

physical generation like the formation of mouse tumors


Infectious material: Three source that underwent PrP depletion

procedures Murine N2a neuroblastoma (22L) Hamster brain (263K) Japanese FU-CJD patient (FU-CJD)

Nucleic acid extractions Usage of Proteinase K for digestion of endogenous or

added nucleases Used purification method for digested genetic material


Ф29 polymerase and ssDNA chromatography Allow for the amplification of circular ssDNA from

the digested nucleic acid RNA and RNAase inhibited the effects of Ф29

polymerase ssDNA was isolated from these factors and dsDNA

before replication Restriction enzyme analysis, PCR and sequencing

Digestion of the Ф29 polymerase product -> PCR proliferation -> sequenced


Demonstration of two new circular DNAs that is in high concentration in TSE particles All three strains tested strongly positive for Sphinx 1.8kb

and 2.4kb, which show a clear viral plasmid structure Both are passed down to daughter cells Both are also present in uninfected brains, but the

concentration in infected brain is x2,500 higher Positive control: Mitochondrial DNA was used

In essence, Sphinx DNA is viral, their actual role is up for further investigation


Viral Detection

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