Advanced

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

Eliasson, Linda LU (2003)
Abstract (Swedish)
Popular Abstract in Swedish

Infektionssjukdomar är lika vanliga hos fiskar som hos alla andra djur. Dessa infektioner kan orsakas av virus, bakterier samt eukaryota organismer som svamp och protozoer. I naturliga fiskpopulationer utgör detta ett litet problem men får stora ekonomiska konsekvenser i fiskodlingar eftersom ett stort antal fiskar kan infekteras på kort tid och i värsta fall dö, ca 10 % av all odlad fisk går förlorad pga. infektionssjukdomar. I detta arbete har struktur och egenskaper hos ett virus, infektiöst pankreatiskt nekrosvirus (IPNV), studerats. Detta virus förorsakar en akut och smittsam sjukdom (infektiös pankreatisk nekros, IPN) hos laxfiskar. Då IPNV infekterar en laxodling kan dödligheten bland... (More)
Popular Abstract in Swedish

Infektionssjukdomar är lika vanliga hos fiskar som hos alla andra djur. Dessa infektioner kan orsakas av virus, bakterier samt eukaryota organismer som svamp och protozoer. I naturliga fiskpopulationer utgör detta ett litet problem men får stora ekonomiska konsekvenser i fiskodlingar eftersom ett stort antal fiskar kan infekteras på kort tid och i värsta fall dö, ca 10 % av all odlad fisk går förlorad pga. infektionssjukdomar. I detta arbete har struktur och egenskaper hos ett virus, infektiöst pankreatiskt nekrosvirus (IPNV), studerats. Detta virus förorsakar en akut och smittsam sjukdom (infektiös pankreatisk nekros, IPN) hos laxfiskar. Då IPNV infekterar en laxodling kan dödligheten bland yngel och småfisk överskrida 50 %. Det är bara yngel och unga fiskar som uppvisar sjukdomssymptom medan vuxna individer blir livslånga bärare och kan föra viruset vidare till sin avkomma samt via vattnet till andra fiskar utan att själva vara märkbart sjuka. Infektionen yttrar sig i att kroppen blir mörkare, buken svullnar upp, ögonen blir utstående och rörelserna ryckiga. Inuti fisken blir lever, hjärta, njurar och mjälte bleka och cellerna i bukspottskörteln (pankreas) dör fläckvis. IPNV kan också infektera andra havslevande djur, t.ex. musslor och kräftdjur, men inga däggdjur. Det är ett generellt problem i fiskodlingar att inte snabbt kunna isolera och identifiera eventuella virus i det vatten som tillförs, finns i eller lämnar odlingen. Under dessa studier har vi konstruerat en apparat och utvecklat en metod för att ur stora vattenvolymer koncentrera och detektera små mängder IPNV. Inom 24 timmar från det att ett vattenprov (5 liter) kommer till laboratoriet kan man med denna metod påvisa eventuell förekomst av IPNV. I alla virussystem är det viktigt att känna till förhållandet mellan det totala antalet viruspartiklar och halten partiklar med förmåga att infektera en cell (specifik infektivitet). Under detta arbete har en metod tagits fram som ger en statistiskt säker bestämning av såväl total som specifik infektivitet. IPNV är ett ikosaederformat virus utan hölje vars arvsmassa består av två dubbelsträngade RNA-segment. IPNV tillhör virusfamiljen Birnaviridae, där namnet syftar på några av virusens kännetecken: bi- står både för de två RNA-segmenten och att RNAt är dubbelsträngat, medan -rna betecknar att arvsmassan består av just RNA. IPNVs kapsid, det yttre proteinskalet på viruspartikeln, är glykosylerad (innehåller olika sockerarter), vilket vi har visat under dessa studier med flera olika metoder och vi har även kunnat visa att ett av virusets interna proteiner är glykosylerat. Vi har vidare demonstrerat att en skillnad föreligger i glykosylering beroende på i vilket cellslag viruset har replikerats. Vi har också producerat och isolerat virusets ytprotein i två former: utan glykosylering (uttryckt i en bakterie) och med glykosylering (framrenat från isolerade och disintegrerade viruspartiklar). Dessa proteiner kommer att användas i studier kring vilken roll glykosyleringen spelar i detta virus-cell system avseende t.ex. receptor interaktionen, huruvida glykosyleringen påverkar immunogenisiteten samt i identifieringsförsök av cellreceptorn för IPNV. (Less)
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... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Linné, Tommy, MBV, SLU, BMC, Uppsala
organization
publishing date
type
Thesis
publication status
published
subject
keywords
bacteriology, virology, mycology, Mikrobiologi, bakteriologi, virologi, mykologi, Microbiology, VP2, Glycosylation, Immuno dot blot, IPNV, TCID50
pages
124 pages
publisher
Linda Eliasson, Department of Cell and Organism Biology, Lund University, Sölvegatan 35, SE- 223 62 Lund, Sweden,
defense location
Lecture hall, Biologybuilding, Sölvegatan 35, Lund
defense date
2003-04-04 10:15
ISBN
91-85067-01-6
language
English
LU publication?
yes
id
c233ad24-eaab-41eb-80d2-38a4bf17edc8 (old id 465544)
date added to LUP
2007-09-04 11:05:44
date last changed
2016-09-19 08:45:07
@misc{c233ad24-eaab-41eb-80d2-38a4bf17edc8,
  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.},
  author       = {Eliasson, Linda},
  isbn         = {91-85067-01-6},
  keyword      = {bacteriology,virology,mycology,Mikrobiologi,bakteriologi,virologi,mykologi,Microbiology,VP2,Glycosylation,Immuno dot blot,IPNV,TCID50},
  language     = {eng},
  pages        = {124},
  publisher    = {ARRAY(0x95025f0)},
  title        = {Infectious Pancreatic Necrosis Virus (IPNV) - Structural Studies and Methodology},
  year         = {2003},
}