Abstract
Despite decades of research efforts, the nature of the infectious agent causing scrapie and other Transmissible Spongiform Encephalopathies (TSE) remains an enigma. The protein-only prion hypothesis posits that an abnormal conformer of a host protein is the infectious agent. Virus and virino theories include host-independent nucleic acids in the genome of the infectious agent, in addition to the protein component (a host protein in the case of virino, and a viral protein in the case of a virus). Viral or sub-viral nucleic acids have long been sought in scrapie to explain the existence of multiple agent strains. Despite a plethora of different approaches to the search, no scrapie-specific nucleic acid sequences have been found in infected cells or tissues. Most viruses induce synthesis of long double stranded RNA (dsRNA) during their replication in cells, and thus the presence of long dsRNA would be an indication of viral infection in cells. J2 monoclonal antibody against long dsRNA is a useful tool for easy screening of cells and tissues for the presence of suspected viral infection; however, this antibody has not previously been used for testing of scrapie infected tissues. Here, we present evidence for long dsRNA in scrapie infected cells and tissues. Such dsRNA is also found in scrapie free tissue culture cells. We believe this may be the first evidence of viral infection in scrapie susceptible and infected cells.
Highlights
Transmissible Spongiform Encephalopathies are rare neurodegenerative brain disorders in both humans (e.g. Creutzfeldt-Jakob disease and Kuru) and animals (Scrapie in sheep, Bovine Spongiform Encephalopathy in cows and Chronic Wasting Disease in deer and elk), characterized by a long incubation period after initial infection
The infectivity of recombinant prion protein misfolded in a test tube in a mixture with RNA and lipid and later injected into the animal brains was demonstrated a few years ago but was never reproduced independently in a laboratory free of contamination[2]
Data presented here shows double stranded RNA (dsRNA) is detectable by J2 antibody using immunofluorescence in scrapie susceptible and scrapie infected tissue culture cells
Summary
Transmissible Spongiform Encephalopathies are rare neurodegenerative brain disorders in both humans (e.g. Creutzfeldt-Jakob disease and Kuru) and animals (Scrapie in sheep, Bovine Spongiform Encephalopathy in cows and Chronic Wasting Disease in deer and elk), characterized by a long incubation period after initial infection. Methods/results Immunofluorescence JFH1 Huh[7] cells (human hepatoma cells harboring hepatitis C replicon) and Huh7.5 cells (human hepatoma cells free of replicon) were used as a positive (JFH1 Huh7) and negative (Huh7.5) control for dsRNA in immunofluorescence detection experiments (Figure 1a) when probing PK1 cells (a clone of mouse neuroblastoma N2A cells) and RML (Rocky Mountain Laboratory strain of mouse scrapie) infected PK1 (RML/PK1) cells with J2 antibody (mouse monoclonal from Englsih and Scientific Consulting) (Figure 1b). In PK1 and RML/PK1 in addition to dsRNA in the upper part of the gel slots several bands were seen including a duplet with a molecular weight much lower than that of RF of HCV replicon (Figure 2). An attempt was made to detect dsRNA in 22L scrapie infected mouse brains fixed in Carnoy’s solution and embedded in paraffin.
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