Characterisation of the genome of Nudaurelia Omega Virus

Abstract

Nudaurelia co virus (Nco V) is a small RNA virus belonging to the Family Tetraviridae. Nco V was successfully isolated from field collected larvae of the pine emperor moth, Nudaurelia cytherea capensis. By polyacrylamide gel electrophoresis-it was possible Jo determine the size of the capsid proteins. Anti-NcoV antiserum was raised by inoculating a rabbit with purified virus. RNA was extracted from the purified virus using a phenolchloroform extraction procedure. It was possible to separate the viral RNA into its constituent species using sucrose density gradient centrifugation. The sizes of both species of RNA was accurately determined by agarose gel electrophoresis. These sizes corresponded to the replicative form of the RNA which was extracted from infected host tissue. The absence of a poly(A) tract on the RNA was shown through poly(U) sepharose chromatography. Cell-free translation of the viral RNA elucidated the sizes of proteins encoded in vitro in a rabbit reticulocyte lysate system. Optimal conditions for in vitro translation of Nco V were determined for a range of conditions. Immunoprecipitaion of viral encoded proteins with anti-Nco V antiserum suggested that the putative coat protein of the virus was encoded by RNA 2, as a precursor polypeptide which underwent posttranslational cleavage. Reverse transcription - polymerase chain reaction (RT -PCR) was used to successfully produce a radiolabelled probe which could detect dot-blotted viral RNA. The efficacy of this probe in detecting the presence of Nco V RNA in infected tissue was also tested.