Journal
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
Volume 81, Issue 4, Pages 1210-1224Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/AEM.03153-14
Keywords
-
Categories
Funding
- Grant Fondecyt (CONICYT-Ministerio de Educacion, Gobierno de Chile) [11110212]
- CONICYT Ph.D. scholarship program
- CONICYT through an Insertion of Human Capital to the Academy [79100014]
- Program Iniciativa Cientifica Milenio from the Chilean Ministry of Economy, Development, and Tourism
Ask authors/readers for more resources
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 6.5 x 10(5) 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
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available