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.
Subject(s)
Isavirus/genetics , Isavirus/physiology , Ribonucleoproteins/biosynthesis , Virus Replication , Animals , Cell Line , DNA-Directed RNA Polymerases/metabolism , Green Fluorescent Proteins/chemistry , Isavirus/isolation & purification , Luciferases/chemistry , RNA , RNA, Viral/genetics , Recombinant Fusion Proteins/biosynthesis , Ribavirin/pharmacology , Ribonucleoproteins/chemistry , Ribonucleoproteins/genetics , Ribonucleoproteins/isolation & purification , Salmo salar/virology , Viral Proteins/geneticsABSTRACT
Vitellogenin is a phosphoglycoprotein which represents the main precursor of the egg yolk in teleost fish. This reproductive protein was also demonstrated to play an important role in innate immunity by acting as a pattern recognition molecule capable of binding to bacteria, fungi and enhancing macrophage phagocytosis. The presented results demonstrate that, egg homogenate, ovarian fluid and serum of mature female Atlantic salmon have high neutralising ability for infectious pancreatic necrosis virus (IPNV). Vitellogenin from mature female Atlantic salmon serum, purified by immuno-affinity on a column matrix coated with monoclonal anti-Atlantic salmon vitellogenin antibody, was able to neutralise between 9.1 x 10(4) and 3.09 x 10(5) TCID(50) IPNV mg(-1) of protein. To the author's knowledge, this is the first time that the neutralising activity of vitellogenin on a teleost virus has been demonstrated. The results may explain why IPNV is difficult to detect by culture methods in ovarian fluid and egg homogenates from carrier mature females and suggest a possible means of vertical transmission via the egg.
