The piscine Orthomyxovirus called Infectious Salmon Anemia Virus (ISAV) is one of the most important emerging pathogens affecting the salmon industry worldwide. The first reverse genetics system for ISAV, which allows the generation of recombinant ISA virus (rISAV), is an important tool for the characterization and study of this fish virus. The plasmid-based reverse genetics system for ISAV includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). The salmon, viral and mammalian genetic elements included in pSS-URG vectors allow the expression of the eight viral RNA segments. In addition to four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex, the eight pSS-URG vectors allowed the generation of infectious rISAV in salmon cells.
Infectious Salmon Anemia Virus ISAV Viral RNA Reverse genetics Salmon cells Transfection ITS-1 RNA polymerase I and RNA polymerase II
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We thank Prof. Daniel R. Perez for technical and scientific support during ISAV reverse genetics development. We are grateful to Dee Sagawe for proofreading the manuscript. We acknowledge financial support Fondecyt 11110212, Fondecyt 1161006, VIU 14E025. M.C.-S.M. thanks to DICYT-USACH and DGT-USACH. D.T.-A. thanks Conicyt-Chile for a PhD fellowship.
Dannevig BH, Falk K, Namork E (1995) Isolation of the causal virus of infectious salmon anaemia (ISA) in a long-term cell line from Atlantic salmon head kidney. J Gen Virol 76(Pt 6):1353–1359CrossRefPubMedGoogle Scholar
Garcia-Sastre A, Palese P (1993) Genetic manipulation of negative-strand RNA virus genomes. Annu Rev Microbiol 47:765–790CrossRefPubMedGoogle Scholar
Hoffmann E, Webster RG (2000) Unidirectional RNA polymerase I-polymerase II transcription system for the generation of influenza A virus from eight plasmids. J Gen Virol 81(Pt 12):2843–2847CrossRefPubMedGoogle Scholar
Castro J et al (1997) Molecular analysis of a NOR site polymorphism in brown trout (Salmo trutta): organization of rDNA intergenic spacers. Genome 40(6):916–922CrossRefPubMedGoogle Scholar
Reed KM, Hackett JD, Phillips RB (2000) Comparative analysis of intra-individual and inter-species DNA sequence variation in salmonid ribosomal DNA cistrons. Gene 249(1–2):115–125CrossRefPubMedGoogle Scholar
Van Herwerden L, Caley MJ, Blair D (2003) Regulatory motifs are present in the ITS1 of some flatworm species. J Exp Zool B Mol Dev Evol 296(1):80–86CrossRefPubMedGoogle Scholar
Toro-Ascuy D et al (2015) 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. Appl Environ Microbiol 81(4):1210–1224CrossRefPubMedPubMedCentralGoogle Scholar
Lanoix J, Acheson NH (1988) A rabbit beta-globin polyadenylation signal directs efficient termination of transcription of polyomavirus DNA. EMBO J 7(8):2515–2522PubMedPubMedCentralGoogle Scholar
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
Castillo-Cerda MT et al (2014) Development of plaque assay for Chilean infectious salmon anaemia virus, application for virus purification and titration in salmon ASK cells. J Fish Dis 37(11):989–995CrossRefPubMedGoogle Scholar
Munir K, Kibenge FS (2004) Detection of infectious salmon anaemia virus by real-time RT-PCR. J Virol Methods 117(1):37–47CrossRefPubMedGoogle Scholar