Infectious Pancreatic Necrosis Virus (IPNV) - Structural Studies and Methodology

Sammanfattning: Infectious pancreatic necrosis virus (IPNV) is a Birnavirus that infects salmonid fish. The infection is usually mortal for fish under six months of age, while older fish become carriers, usually without any signs of infection, and will spread the virus to other susceptible fish and to their offspring. IPNV is an icosahedral, naked virion and the genome consists of two segments of double-stranded RNA. The virus genome codes for five proteins, three of which are structural components. The virus protein 2 (VP2) is the major protein component of the capsid and it also functions as the virus attachment protein (VAP) of IPNV. In this study we have used three different methods to prove that VP2 is glycosylated: lectin blots, metabolic radiolabelling and gas chromatography-mass spectrometry. We showed that VP2 carries mannose, glucose and galactose. We have further seen a quantitative difference in glycosylation depending on in which cell line the virus has been propagated. During these studies glycosylation of the internal protein VP1, has been shown and this protein is found in the virion both in a free form and as a genome-linked protein. We have also produced and purified recombinant VP2 in its unglycosylated form from bacteria, and isolated and purified authentic, glycosylated VP2 from disintegrated virions. To study the possible significance of virion glycosylation these proteins are to be used in attachment and infectivity competition experiments with infectious viruses in cell cultures. The two variants of VP2 are presently used to produce monospecific antibodies, which will be used in studies on the mechanisms of antibody-mediated neutralization. For infectivity titrations of IPNV an immuno dot blot TCID50 assay, was developed which measures virions and viral proteins in the growth medium of infected cells. The method gives clear cut answers if the cells are virus-infected or not, thus abolishing the need for otherwise subjectively estimated limits. This method was used in a study of different storage conditions for IPNV, and the best storage condition was found to be at -70°C with glycerol as a cryoprotective agent. In the aquaculture industry it can be a problem to detect and identify viruses in the water entering or leaving the rearing facilities. In this study we have developed a method and an apparatus that easy and efficiently will concentrate IPNV from large water volumes by a factor of at the most 250,000 times. Within 24 hrs the viruses will be detected and identified by serological methods.