title: Development of a reverse genetic system for infectious salmon anemia virus: rescue of recombinant fluorescent virus by using salmon internal transcribed spacer region 1 as a novel promoter.
abstract: Infectious salmon anemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), belonging to the genus Isavirus, family Orthomyxoviridae. There is an urgent need to understand the virulence factors and pathogenic mechanisms of ISAV and to develop new vaccine approaches. Using a recombinant molecular biology approach, we report the development of a plasmid-based reverse genetic system for ISAV, which includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). Salmon cells cotransfected with pSS-URG-based vectors expressing the eight viral RNA segments and four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex allowed the generation of infectious recombinant ISAV (rISAV). We generated three recombinant viruses, wild-type rISAV(901_09) and rISAVr(S6-NotI-HPR) containing a NotI restriction site and rISAV(S6/EGFP-HPR) harboring the open reading frame of enhanced green fluorescent protein (EGFP), both within the highly polymorphic region (HPR) of segment 6. All rescued viruses showed replication activity and cytopathic effect in Atlantic salmon kidney-infected cells. The fluorescent recombinant viruses also showed a characteristic cytopathic effect in salmon cells, and the viruses replicated to a titer of 6.5105 PFU/ml,similar to that of the wild-type virus. This novel reverse genetics system offers a powerful tool to study the molecular biology of ISAV and to develop a new generation of ISAV vaccines to prevent and mitigate ISAV infection, which has had a profound effect on the salmon industry.
authors.forename: Daniela
authors.initials: D
authors.lastname: Toro-Ascuy
authors.forename: Carolina
authors.initials: C
authors.lastname: Tambley
authors.forename: Carolina
authors.initials: C
authors.lastname: Beltran
authors.forename: Carolina
authors.initials: C
authors.lastname: Mascayano
authors.forename: Nicolas
authors.initials: N
authors.lastname: Sandoval
authors.forename: Eduardo
authors.initials: E
authors.lastname: Olivares
authors.forename: Rafael A
authors.initials: RA
authors.lastname: Medina
authors.forename: Eugenio
authors.initials: E
authors.lastname: Spencer
authors.forename: Marcelo
authors.initials: M
authors.lastname: Cortez-San Martín
mesh_terms: D000818:Animals; D005393:Fish Diseases / Q000821:virology*; D029941:Fish Proteins / Q000235:genetics* / Q000378:metabolism; D005453:Fluorescence; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D046829:Isavirus / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D009976:Orthomyxoviridae Infections / Q000662:veterinary* / Q000821:virology; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D059386:Reverse Genetics / Q000379:methods*; D020317:Salmo salar / Q000821:virology; D014779:Virus Replication
chemical_list: D029941:Fish Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins