Molecular interaction of infectious salmon anaemia virus and the Atlantic salmon innate immune system

Infectious salmon anaemia (ISA) is a fatal viral disease of Atlantic salmon. Despite more than two decades of research to provide knowledge for instituting effective control measures, the disease continues to cause devastating losses, most recently in Chile and Scotland. Research aimed at better understanding the initial stages of the virus-host cell interactions is required to generate more knowledge on the pathogenesis and immunology of the disease process. The thesis project looked into the molecular interaction of ISA virus (ISAV) and the Atlantic salmon cells (erythrocytes, Chinook salmon embryo CHSE-214 cells, and Atlantic salmon TO macrophage/dendritic-like cells). Transmission electron microscopy used to examine the physical interaction between ISAV and erythrocytes provided evidence that ISAV undergoes endocytosis in Atlantic salmon erythrocytes. A follow-up study examined the possibility of ISAV replication and expression of type I interferon (IFN) system genes in Atlantic salmon erythrocytes following ISAV haemagglutination. Haemagglutination induced by the high pathogenicity isolate NBISA01 but not the low pathogenicity isolate RPC/NB-04-0851 resulted in productive infection as evidenced by increased ISAV segment 8 transcripts and increase in the median tissue culture infectious dose (TCID50). Moreover, ISAV up-regulated the expression of the mRNA levels of key type I IFN system genes (IFN-alpha, Mx, ISG15, STAT1) in erythrocytes. Although Atlantic salmon TO cells are known to up-regulate the expression of type I IFN system genes, information on the effect of ISAV strain variation on this expression is lacking. To better understand this interaction, the two ISAV isolates of differing pathogenicity phenotype and genotypes (NBISA01 and RPC/NB-04-085-1) were initially used to infect TO and CHSE-214 cells and the mRNA levels of key type I IFN system genes and ISAV transcripts were measured by real-time quantitative reverse transcription-Polymerase chain reaction (QRT-PCR). The results of the TO cell experiment showed remarkable differences in the expression of the key type I IFN system genes and viral transcripts in TO cells in response to the two ISAV isolates. NBISA01 replicated robustly and showed very low mRNA levels of the key type I IFN system genes. On the other hand, RPC/NB-04-085-1 replicated slowly and showed higher mRNA levels of the type I IFN system genes. Based on these results, we proceeded to characterize the Atlantic salmon TO cell global gene expression responses to infection with NBISA01 and RPC/NB-04-085-1 using microarray analysis and validation by QRT-PCR. Overall, the microarray experiment showed that RPC/NB-04-085-1-infected cells had a higher total number of reproducibly dysregulated genes than the NBISA01-infected cells. The 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. A further study was carried out to expand the knowledge Oil the expression of microarray identified immune response genes using a selection of 4 ISAV isolates (NBISA01, RPC/NB-04-085-1, RPC/NB-0593-1, and Norway-810/9/99) that differ in pathogenicity and geographic origins. The RPC/NB-04-085-1 infected cells showed the highest mRNA expression for most immune-relevant genes, followed by Norway-810/ 9/99. NBISA01 and RPC/NB-01-0593-01 (both of North American genotype) showed lower mRNA expression of the genes that were highly expressed by RPC/NB-04-085-1 infected cells. These findings show that ISAV isolates have strain-specific variations in their ability to induce fish immune response genes.