title: Development of infectious cDNA clones of Salmonid alphavirus subtype 3.
abstract: BACKGROUND
  Salmonid alphavirus (SAV) is a widespread pathogen in European aquaculture of salmonid fish. Distinct viral subtypes have been suggested based on sequence comparisons and some of these have different geographical distributions. In Norway, only SAV subtype 3 have so far been identified. Little is known about viral mechanisms important for pathogenesis and transmission. Tools for detailed exploration of SAV genomes are therefore needed.
  
  
  RESULTS
  Infectious cDNA clones in which a genome of subtype 3 SAV is under the control of a CMV promoter were constructed. The clones were designed to express proteins that are putatively identical to those previously reported for the SAVH20/03 strain. A polyclonal antiserum was raised against a part of the E2 glycoprotein in order to detect expression of the subgenomic open reading frame (ORF) encoding structural viral proteins. Transfection of the cDNA clone revealed the expression of the E2 protein by IFAT, and in serial passages of the supernatant the presence of infectious recombinant virus was confirmed through RT-PCR, IFAT and the development of a cytopathic effect similar to that seen during infection with wild type SAV. Confirmation that the recovered virus originated from the infectious plasmid was done by sequence identification of an introduced genetic tag. The recombinant virus was infectious also when an additional ORF encoding an EGFP reporter gene under the control of a second subgenomic alphavirus promoter was added. Finally, we used the system to study the effect of selected point mutations on infectivity in Chinook salmon embryo cells. While introduced mutations in nsP2197, nsP3263 and nsP3323 severely reduced infectivity, a serine to proline mutation in E2206 appeared to enhance the virus titer production.
  
  
  CONCLUSIONS
  We have constructed infectious clones for SAV based on a subtype 3 genome. The clones may serve as a platform for further functional studies.
authors.affiliation: Department of Biology, University of Bergen, Thor Møhlens gate 55, 5020 Bergen, Norway. marius.karlsen@bio.uib.no.
authors.forename: Marius
authors.initials: M
authors.lastname: Karlsen
authors.forename: Stephane
authors.initials: S
authors.lastname: Villoing
authors.forename: Karl F
authors.initials: KF
authors.lastname: Ottem
authors.forename: Espen
authors.initials: E
authors.lastname: Rimstad
authors.forename: Are
authors.initials: A
authors.lastname: Nylund