Techniques of RNA Isolation and Characterization in Infectious Pancreatic Necrosis Virus-Infected Chinook Salmon Embryo Cells
Infectious pancreatic necrosis virus (IPNV) causes the most lethal disease in young salmonid fishes with mortality rates of equal or higher than ninety-five percent. The survival of the adult fish has been the main cause of this virus's proliferation around the world, making it the most economically devastating viral disease for fish hatcheries. This prototype naked, double-stranded RNA virus of the Birnaviradae family was studied to test new techniques for isolation and characterization of its ambiguous early post-infection viral RNA products. RNA was extracted by either the classical phenol method or a new kit prepared by Qiagen Rneasy Mini Kit. The Qiagen kit was compared for efficacy and it proved superior to the standard phenol extraction technique. A viral genomic RNA probe was labeled with psoralen-biotin and used in an attempt to detect virus-specific RNA species at designated post-infection times. Detection involved separating RNA on formaldehyde denaturing gels, northern transfer and in situ hybridization. The results confirmed the previously published data on the release time of the virus and highlighted the difficulties in mass virus and viral RNA production, which limited the capabilities of these complex techniques.
Networked Digital Library of Theses and Dissertations