Salmonid alphaviruses (SAV) are a group of viruses that have recently emerged as a serious threat to the salmonid aquaculture industry in Europe. Over recent years, diseases caused by SAV have severely hampered the Scottish, Irish and Norwegian Atlantic salmon industry, and are considered to be among the major economically important viral diseases affecting the industry at present. Amongst the six subtypes characterised so far, Salmonid alphavirus 1 (SAV1) causes severe pathology in the heart, pancreas and the skeletal muscle of Atlantic salmon leading to death and growth retardation in the affected fish. The biochemical characteristics of the virus and the sequential pathology of the diseases caused by SAV have been described; however the mechanisms responsible for causing the disease and the host defence mechanisms against the virus are poorly defined. This thesis therefore examined the pathogenesis of SAV infection at the cellular and molecular level in vivo in salmon and in vitro in salmonid cells, with a special emphasis on host immune defence mechanisms against the virus. SAV was first isolated from Chinook salmon embryo-214 (CHSE-214) cells in 1995 in Ireland. Several cell lines have since been used to grow the virus. In the present study, three established salmonid cell lines, Chum salmon heart -1 (CHH-1), CHSE-214 and Salmon head kidney -1 (SHK-1) were evaluated for their ability to support the isolation of SAV-1 from infected fish tissue, with CHH-1 cells giving the fastest cytopathic effect (CPE) during primary isolation. The CPE appeared as localised cell-rounding on CHH-1 and CHSE-214 cells, although in SHK-1 cells, the cells were seen to slough off the monolayer relatively later than with the other two cell lines during the infection. The host response to SAV infection was evaluated by experimentally infecting Atlantic salmon parr using a cell culture-adapted virus isolate. A quantitative reverse transcription polymerase chain reaction (qRT-PCR) was developed to examine the virus load in the fish, from which it was found that the highest viral RNA copy number was detected at 5 day post infection (d.p.i), of the 90 day experimental infection period. Characteristic pathological lesions were only seen in the pancreas and the heart but not in the skeletal muscles of the infected fish. A gene expression study using qRT-PCR revealed the rapid induction of interferon (INF) and INF-associated genes in the head kidney of the infected fish compared to the control fish. The Mx protein was found to be highly expressed in the heart and the mucous membranes of infected fish by immunohistochemistry. Interestingly, the pathological changes that were seen occurred some time after the peak expression of genes associated with the INF-1-pathway. When the host-virus interaction of Atlantic salmon infected with SAV was examined using a microarray, a potent first line defence response was observed, together with the signatures of early activation of the adaptive immune response during the initial stages of the infection. Genes associated with transcription, translation and lipid metabolism were significantly differentially expressed in virus infected fish compared to control fish. A large array of antiviral genes was significantly expressed, amongst which were some of the genes also described in mammalian alphavirus infections. Genes associated with apoptosis and anti-apoptosis were also seen to be differentially regulated showing the complexity of the host-virus interaction. Collectively, all of these findings suggest that a non-specific antiviral immune response takes place providing rapid immune protection during the early stages of SAV infection in salmon. In the study on morphogenesis of SAV in salmonid cells using electron microscopy (EM), a rapid internalization of virus into the cells and generation of replication complexes using the secretory pathway of the cell, similar to mammalian alphavirus replication was observed. The mature viruses were released through surface projections, acquiring envelopes from the host cell membrane. From the ultrastructural studies of the salmonid cells infected with SAV, a progressive chromatin marginalisation and condensation could be seen, leading to cellular fragmentation, forming membrane bound apoptotic bodies, characteristic of progressive apoptosis. The activation of caspase-3 in the cytoplasm and genomic DNA damage were also seen in the infected fish cells, indicating that apoptosis is the main cause of cell death during SAV infection. The results of this study have increased our knowledge and understanding of the cellular and molecular mechanisms involved in the pathogenesis of SAV infection, emphasising the importance of the first line defence mechanisms against SAV infection in salmon. This has given an interesting insight into the host mechanisms used to combat the virus during infection, and will undoubtedly be useful for designing new vaccines and management strategies for prevention and control of this important disease

Over the past few years one of the major concerns in the Atlantic salmon (Salmo salar) farming industry has been the increasing incidence and severity of inflammatory viral diseases. Heart and skeletal muscle inflammation (HSMI) and cardiomyopathy syndrome (CMS) are currently two of the most prevalent viral diseases in commercial Atlantic salmon farms in Norway. Mortality levels in both diseases are generally low but morbidity can be very high with the associated chronic inflammatory response lasting for several months. The consequent reduced growth performance is causing considerable financial impact as HSMI has become increasingly widespread in recent years. The impact of CMS is further exacerbated as it generally affects large fish close to harvest. HSMI lesions occur in the atrium and ventricle in the heart including inflammation and necrosis in epi- endo- and myocardium along with myositis of red skeletal muscle. CMS lesions are commonly observed in the spongy myocardium in the atrium and ventricle of the heart with severe mononuclear inflammation and necrosis. Furthermore, circulatory disturbances associated with reduced cardiac function cause multifocal liver steatosis and necrosis in both diseases. Currently there are no vaccines or any other effective treatments for these diseases and so alternative therapies that could potentially modulate the intensity of the inflammatory response could be crucial to improve the clinical manifestation of the diseases. Therefore, the overall aim of the present study was to evaluate the concept of “clinical nutrition” to improve the clinical symptoms of both viral diseases, HSMI and CMS, through the use of functional feeds formulated with reduced lipid content and increased proportions of anti-inflammatory fatty acids to moderate the apparently uncontrolled inflammatory response in the heart tissue associated with both diseases and also alleviate the secondary hepatic lesions. The experimental work consisted of three major dietary trials in Atlantic salmon in seawater. Two large trials investigated the effects of functional feeds in Atlantic salmon challenged with Atlantic salmon piscine reovirus (ASRV) and piscine myocarditis virus (PMCV), the causal agents of HSMI and CMS, respectively. In both trials, heart transcriptome, heart and liver histopathology and tissue lipid and fatty acid compositions and metabolism were determined post-infection in fish fed with the functional feeds in comparison with fish fed with a standard commercial feed formulation considered as a reference diet. All the functional feeds were formulated to have reduced digestible energy through lower dietary lipid and higher protein contents, and increased levels and proportions of anti-inflammatory long-chain polyunsaturated fatty acids (LC-PUFA), particularly eicosapentaenoic acid (EPA) compared with the reference diets. Histopathology, fatty acid composition and gene expression of heart were assessed over a long time-period of 16 weeks and 14 weeks post-challenge with ASRV and PMCV, respectively. Viral load in heart tissue, hepatic histopathology and fatty acid composition of liver and head kidney along with expression of the genes involved in the eicosanoid and LC-PUFA and eicosanoid biosynthesis pathways were also determined in the HSMI trial. The third trial was a nutritional trial evaluating the effects of dietary digestible energy content on lipid and fatty acid metabolism in salmon fed diets containing graded amounts of lipid. Fatty acid composition of liver and heart were assessed over 12 weeks, along with the hepatic expression of genes of lipid and fatty acid metabolism. The results of this research are presented in four chapters (Chapters 2-5) as four paper manuscripts. The manuscripts/Papers are either published (Chapter 2), in review (Chapter 3 and 4) or drafted for submission (Chapter 5) in appropriate peer-reviewed international journals. Chapter 2 and 3 correspond to the HSMI trial, Chapter 4 to the nutritional trial, and Chapter 5 to the CMS trial. Chapter 2 showed that viral load and histopathology scores were lower in fish fed the functional feeds, especially diet FF1, which displayed better performance. Diet strongly influenced the expression of genes related with the immune and inflammatory responses, with delayed expression in fish fed the functional feeds. Up-regulation of pro-inflammatory genes was correlated with the higher viral load observed at early-mid stages of the disease in fish fed the reference diet (ST). Expression of genes related with the immune response at 16-weeks post challenge reflected the differences in immunomodulation between the functional feeds, with fish fed diet FF1 showing lower expression. Therefore, severity of the heart lesions was correlated with the intensity of the immune response and could be associated with tissue anti-inflammatory LC-PUFA levels. Chapter 3 was focused on liver histopathology, fatty acid composition and LC-PUFA biosynthesis, along with phospholipid fatty acid composition and eicosanoid production in head kidney and heart tissue at early and late stages of ASRV infection. Liver was severely affected by the virus at the beginning of the infection in fish fed the reference ST diet, but the level of lesions were similar in all dietary groups at the end of the trial. Hepatic expression of fatty acyl desaturases was significantly depressed in fish fed the ST diet compare with fish fed the functional feeds despite the lower levels of dietary LC-PUFA in that feed. Thus endogenous production and bioavailability of anti-inflammatory LC-PUFA was potentially enhanced in fish fed the functional feeds. Changes in tissue lipid content, mobilization of fatty acids involved in inflammatory responses and changes in expression of transcription factors and genes involved in eicosanoid biosynthesis were more prominent in head kidney, confirming the important role of this organ in dietary immunomodulation after viral infection. To a lesser extent similar changes were observed in heart tissue, suggesting in situ production of eicosanoids could also be important. The unexpected effects of diet on expression of genes of LC-PUFA biosynthesis were specifically investigated in the trial described in Chapter 4. One aim of this study was to clarify whether dietary lipid content or viral infection was the cause of altered expression of desaturase genes between the different diets. Hepatic expression of other genes of lipid and fatty acid metabolism were also determined to evaluate metabolic changes associated with dietary lipid/energy level. In general, reduction of dietary energy and lipid contents while maintaining similar proportions of dietary fatty acids, led to a general up-regulation of genes involved in lipid biosynthetic pathways. Thus salmon fed lower energy diet showed increased liver expression of fatty acyl desaturases in comparison with fish fed higher energy levels. Heart transcriptomic data in Chapter 5 showed a similar delay in the inflammatory response in fish fed the functional feeds after PCMV infection as observed in the HSMI study. Modulation of inflammatory responses, similar to that previously described after ASRV infection, was also observed in fish fed the functional feeds. However, the differences in the expression of immune related genes and the level of heart lesions were not as prominent at mid-late stages of the disease as in fish fed FF1 in the HSMI trial. The present study demonstrated the beneficial effects of a clinical nutrition approach via functional feeds in two viral inflammatory diseases, HSMI and CMS, currently affecting farmed Atlantic salmon.

A series of experiments with Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV) were carried out to determine dynamics of the spread of infection in chinook salmon (1.2-1.98g) and rainbow trout (1.2-3.1g). It was found in experiments with A. salmonicida that fish infected by bath immersion became infectious at 4 days postexposure (dpe), one day prior to dying from furunculosis. In cohabitation experiments with a single infected fish donor, an average of 75% disease specific mortality was obtained. There was suggestive evidence that there is a positive relationship between holding volumes and furunculosis prevalence in cohabitation experiments with single donor fish. Median day to infection was inversely correlated with density. The threshold density at density of 1.97 fish/L was approximately 30 times less than the density of 0.08 fish/L, 13.33 and 320 fish respectively. Reproductive ratio (R₀) and transmission coefficient (β) in the furunculosis epizootic were 3.23 and 0.021 (individuals*day)⁻¹ respectively. The mortality rate (α) of infected animals was 28.7% per day. The models constructed successfully mirrored the results of laboratory experiments. Data produced by simulation of the models were significantly associated with the data obtained from laboratory experiments for susceptible (S) class and also for infected class. In similar experiments carried out with IHNV, it was found that donor fish became infectious 3 dpe. The virus levels in donor fish and prevalence levels were also highly associated. Smaller volumes of that led to higher prevalence levels than observed in bigger volumes with 60 or 30 fish in each. A single donor fish was able to transfer virus to recipient fish. However, unlike the A. salmonicida experiment, transmission was insufficient to initiate a full-scale infectious hematopoietic (IHN) epizootic. Estimated parameters for dynamics of infection were approximately half of the values for A. salmonicida (R₀=2.57,β=0.008 (individuals*day)⁻¹ and α=0.15). The models constructed for IHNV spread were used to simulate the results of density experiment. However, it was not possible to test the association between susceptible and infected classes due to inadequate number of infected fish. / Graduation date: 2001