Molecular Immunology 46 (2009) 2955–2974 Contents lists available at ScienceDirect Molecular Immunology journal homepage: www.elsevier.com/locate/molimm Infectious salmon anaemia virus (ISAV) isolates induce distinct gene expression responses in the Atlantic salmon (Salmo salar) macrophage/dendritic-like cell line TO, assessed using genomic techniques Samuel T. Workenhe a , Tiago S. Hori b , Matthew L. Rise b , Molly J.T. Kibenge a , Frederick S.B. Kibenge a,∗ a Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, P.E.I., Canada C1A 4P3 b Ocean Sciences Centre, Memorial University of Newfoundland, 1 Marine Lab Road, St. John’s, NL, Canada A1C 5S7 article info Article history: Received 2 January 2009 Received in revised form 12 June 2009 Accepted 18 June 2009 Available online 18 July 2009 Keywords: ISAV Atlantic salmon Microarray QRT-PCR Fish innate immune responses Virus pathogenicity abstract Infectious salmon anaemia virus (ISAV) is a marine orthomyxovirus of significant interest not only as a cause of a fatal disease of farmed Atlantic salmon resulting in severe economic losses to the aqua- culture industry, but also as the only poikilothermic orthomyxovirus. ISAV targets vascular endothelial cells and macrophages, and is known to influence the expression of both innate and adaptive immune response relevant genes. ISAV isolates from different geographic regions have been shown to vary con- siderably in their pathogenicity for Atlantic salmon. This study aimed to characterize the Atlantic salmon TO macrophage/dendritic-like cell responses to infection with a selection of ISAV isolates of different genotypes and pathogenicity phenotypes. The first TO infection trial used ISAV isolates NBISA01 and RPC/NB-04-085-1 of high and low pathogenicity, respectively, and global gene expression analyses were carried out using ∼16,000 gene (16K) salmonid cDNA microarrays to compare RNA samples extracted from TO cells harvested 24 and 72h post-infection versus time-matched uninfected controls. Overall, the microarray experiment showed that RPC/NB-04-085-1-infected cells had a higher total number of reproducibly dysregulated genes (88 genes: the sum of genes greater than 2-fold up- or down-regulated in all four replicate microarrays of a given comparison) than the NBISA01-infected cells (10 genes) for the combined sampling points (i.e. 24 and 72h). This microarray experiment identified several salmon genes that were differentially regulated by NBISA01 and RPC/NB-04-085-1, and which may be useful as molecular biomarkers of ISAV infection. An initial quantitative reverse transcription-polymerase chain reaction (QRT-PCR) study involving 25 microarray-identified genes confirmed the differences in the level of dysregulation of host transcripts between the two ISAV isolates (i.e. NBISA01 and RPC/NB-04-085-1). A second TO infection trial was run using a selection of four clinical ISAV isolates (Norway-810/9/99, a high pathogenicity isolate of European genotype; RPC/NB-04-085-1, a low pathogenicity isolate of European genotype; NBISA01, a high pathogenicity isolate of North American genotype; and RPC/NB-01-0593-1, an intermediate pathogenicity isolate of North American genotype), and UV-inactivated RPC/NB-04-085-1, with sampling at 24, 36, 48, 72, 96, and 120 h post-infection. The microarray-identified, QRT-PCR validated suite of 24 molecular biomarkers of response to ISAV were used in a second QRT-PCR experiment to assess the TO cell gene expression responses to the four ISAV isolates at all six time points in the infection. The QRT-PCR data showed that RPC/NB-04-085-1 caused the highest fold changes of most immune-relevant genes [such as interferon-inducible protein Gig1, Mx1 protein, interferon-induced protein with tetratri- copeptide repeats 5, Radical S-adenosyl methionine domain-containing protein (viperin), and several genes involved in the ISGylation pathway], followed by Norway-810/9/99. NBISA01 and RPC/NB-01- 0593-01 (both of North American genotype) showed low fold up-regulation of transcripts that were highly induced by RPC/NB-04-085-1 isolate. These findings show that ISAV isolates have strain-specific variations in their ability to induce immune response genes. © 2009 Elsevier Ltd. All rights reserved. ∗ Corresponding author. Tel.: +1 902 566 0967; fax: +1 902 566 0851. E-mail address: kibenge@upei.ca (F.S.B. Kibenge). 1. Introduction Infectious salmon anaemia (ISA) virus (ISAV) is a significant fish pathogen that continues to cause severe economic losses to the salmon-farming industry in an increasing number of countries (Kibenge et al., 2004). The clinical disease, ISA, in marine-farmed 0161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2009.06.015