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

Research output: ThesisDoctoral Thesis (compilation)

Abstract

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.
Original languageEnglish
QualificationDoctor
Awarding Institution
  • Department of Biology
Supervisors/Advisors
  • Everitt, Einar, Supervisor
Award date2003 Apr 4
Place of PublicationLund
Publisher
ISBN (Print)91-85067-01-6
Publication statusPublished - 2003

Bibliographical note

Defence details

Date: 2003-04-04
Time: 10:15
Place: Lecture hall, Biologybuilding, Sölvegatan 35, Lund

External reviewer(s)

Name: Linné, Tommy
Title: [unknown]
Affiliation: MBV, SLU, BMC, Uppsala

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Article: I) Linda Svensson*, Anna Hjalmarsson and Einar Everitt (1999).TCID50 determination by an immuno dot blot assay as exemplified in a study of storage conditions for IPNV.Journal of Virological Methods 80:17-24.

Article: II) Linda Eliasson, Karol Bal, Lennart Larsson and Einar Everitt (2002).Establishment of IPN virion glycosylation by chemical, biological and physiological means.Archives of Virology. Submitted.

Article: III) Linda Eliasson and Einar Everitt.Production and purification of recombinant VP2 produced in Escherichia coli and authentic VP2 derived from disintegrated virions of IPNV.In manuscript.

Article: IV) Linda Eliasson, Stefan Sydoff and Einar Everitt (2002).Capturing ion exchanger-bound infectious pancreatic necrosis virus (IPNV) – design and application for large volume water samples.Journal of Virological Methods. Accepted.

Article: *Paper I was published under the maiden name of Svensson.



The information about affiliations in this record was updated in December 2015.
The record was previously connected to the following departments: Department of Cell and Organism Biology (Closed 2011.) (011002100), Neuronano Research Center (NRC) (013210020), Neurophysiology (013212004)

Subject classification (UKÄ)

  • Microbiology

Free keywords

  • bacteriology
  • virology
  • mycology
  • Mikrobiologi
  • bakteriologi
  • virologi
  • mykologi
  • Microbiology
  • VP2
  • Glycosylation
  • Immuno dot blot
  • IPNV
  • TCID50

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