|Appears in Collections:||Aquaculture eTheses|
|Title:||Cellular and Molecular Pathogenesis of Salmonid Alphavirus 1 in Atlantic Salmon Salmo salar L.|
|Authors:||Herath, Tharangani K.|
|Supervisor(s):||Richards, Randolph Harvey|
Thompson, Kimberly Dawn
Cellular and Molecular Pathogenesis
|Publisher:||University of Stirling|
|Citation:||T K. Herath, J. Z. Costa, K.D. Thompson, A. Adams and R. H. Richards (2009). Alternative Cell Lines for the Isolation of Salmon Alphavirus-1. Icelandic Agricultural Sciences (22)19-27.|
|Abstract:||Abstract 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|
|Type:||Thesis or Dissertation|
|Affiliation:||School of Natural Sciences|
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