MOLECULAR INTERACTION OF INFECTIOUS SALMON ANAEMIA VIRUS AND THE ATLANTIC SALMON INNATE IMMUNE SYSTEM A Thesis Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department of Pathology and Microbiology Faculty of Veterinary Medicine University of Prince Edward Island Samuel Workenhe August 2009 ©2009. 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Copying or publication or any other use of the thesis for financial gain without approval by the University of Prince Edward Island and the author's written permission is prohibited. Requests for permission to copy or make any other use of material in this thesis in whole or in part should be addressed to: Chair of the Department of Faculty of Veterinary Medicine University of Prince Edward Island Charlottetown, PEI Canada CI A4P3 1 SIGNATURE PAGE (n)4(iif) REMOVED ACKNOWLEDGEMENTS This work was supported by a Natural Sciences and Engineering Research Council (NSERC) of Canada Discovery grant to Dr. Frederick Kibenge. I would also like to thank the Department of Pathology and Microbiology, AVC, UPEI for partial stipend top-ups. First and foremost I am very thankful to Dr. Frederick Kibenge for giving me the opportunity to work with him and for his excellent supervision. Dr. Kibenge has been helpful in guiding me the way to be an independent scientist. Many thanks are due to my supervisory committee members, Dr. Glenda Wright, Dr. Edan Foley, Dr. Dave Groman, and Dr. Gerald Johnson, for their best intellectual inputs in my research project and reading the thesis. My acknowledgments extend to Dr. Molly Kibenge for her best mentorship and wise advice in hard times. I would also like to thank Dr. Molly Kibenge, Dr. Matthew Rise, Tiago Hori, Dr. Glenda Wright, Dorota Wadowska, Dr. Dave Groman, and Dr. Tokinori Iwamoto, for their contribution as co-authors of the manuscripts we published. I am highly indebted to my families especially Tinsae, Tigist, and Addis for their significant moral support during my study. Last but not least, my gratefulness is for my friends Wondafrash Eshete, Tizita Wondafrash, and Nina Molla for the fun and caring we shared. IV ABSTRACT 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-a, 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 v 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 IIFN 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 on 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 vi 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. vn ABBREVIATION Ml Microliter HM Micromolar API activator protein 1 APC antigen presenting cells ASK Atlantic salmon kidney ATF-2 activating transcription factor 2 CAB Carassius auratus blastulae CARD caspase recruitment domain CARDIF CARD adaptor inducing IFN-P cDNA complementary DNA CHSE chinook salmon embryo cells CID a central interacting domain CPE cytopathic effect CpG nucleotides cytosine and guanine in repetition CREB cAMP response element binding protein cRNA complementary RNA Ct cycle threshold CTL cytotoxic T lymphocytes DAI DNA-dependent activator of IFN-regulatory factors DC dendritic cells DNA deoxyribonucleic acid dsRNA double stranded RNA EDTA ethylenediaminetetraacetic acid EIF eukaryotic translation initiation factor ELISA enzyme linked immunosorbent assay EST expressed sequence tags F fusion protein FBS fetal bovine serum GAS gamma- associated site GCHV grass carp hemorrhagic septicemia virus GBP guanylate binding protein GED GTPase effector domain HA haemagglutinin protein HE haemagglutinin-esterase HMEM Hanks' minimum essential medium HPR highly polymorphic region IFAT indirect fluorescent antibody test IFN Interferon IKK