title: Production of infectious salmon anaemia virus (ISAV) ribonucleoprotein complexes using a mammalian cell based minigenome system.
abstract: Developments in recombinant virus techniques have been crucial to understand the mechanisms of virulence acquisition and study the replication of many different negatively stranded RNA viruses. However, such technology has been lacking for infectious salmon anaemia virus (ISAV) until recently. This was due in part to the lack of a Polymerase I promoter in Atlantic salmon to drive the production of recombinant vRNA. Therefore, the present study investigated a different alternative to produce ISAV recombinant vRNA, based on Mouse Pol I promoter/terminator sequences and expression in baby hamster kidney (BHK-21) cells. As a first step, a pathogenic ISAV was demonstrated to replicate and produce viable virions in BHK-21 cells. This indicated that the virus could use the mammalian cellular and nuclear machinery to produce vRNA segments and viral proteins, albeit in a limited capacity. Co-transfection of vRNA expressing plasmids with cytomegalovirus (CMV) promoter constructs coding for the three viral polymerase and nucleoprotein led to the generation of functional ribonucleoproteins (RNPs) which expressed either, green fluorescence protein (GFP) or firefly luciferase (FF). Further experiments demonstrated that a 21h incubation at 37°C was optimal for RNPs production. Inhibition by ribavirin confirmed that FF expression was linked to specific RNPs polymerase transcription. The present minigenome system provides a novel and alternative approach to investigate various aspects of ISAV replication and potentially those of other negatively stranded RNA viruses. Expression of RNPs in mammalian cells could also provide a method for the rapid screening of anti-viral compounds targeting ISAV replication.
authors.affiliation: Marine Scotland Science, Marine Laboratory, AB11 9DB, Aberdeen, United Kingdom. Electronic address: Mickael.Fourrier@gov.scot.
authors.forename: Mickael C S
authors.initials: MCS
authors.lastname: Fourrier
authors.affiliation: Marine Scotland Science, Marine Laboratory, AB11 9DB, Aberdeen, United Kingdom. Electronic address: Bertrand.Collet@gov.scot.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Collet
mesh_terms: D000818:Animals; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry; D046829:Isavirus / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology*; D008156:Luciferases / Q000737:chemistry; D012313:RNA; D012367:RNA, Viral / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012254:Ribavirin / Q000494:pharmacology; D012261:Ribonucleoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020317:Salmo salar / Q000821:virology; D014764:Viral Proteins / Q000235:genetics; D014779:Virus Replication
chemical_list: D012367:RNA, Viral; C039919:RNA, recombinant; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D012254:Ribavirin; D012313:RNA; D008156:Luciferases; D012321:DNA-Directed RNA Polymerases
keywords: BHK-21 cells; Infectious salmon anaemia virus; Minigenome; Mouse polymerase I promoter