Cellular and Molecular Pathogenesis of Salmonid Alphavirus 1 in Atlantic Salmon Salmo salar L.

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 diseas

Alternative cell line for the isolation of salmonid alphavirus-1

Salmonid alphavirus (SAV) has recently become an economically important pathogen in salmonid aquaculture in Europe. Subtype SAV-1 causes salmon pancreas disease (SPD) in Atlantic salmon in Scotland and Ireland, and was first isolated on Chinook salmon embryo-214 (CHSE-214) cells in 1995 in Ireland; several established cell lines have since been tested for viral growth, although the ability of these cell lines to support primary virus isolation has not being examined. In the present study, CHSE-214, Chum salmon heart -1 (CHH-1) and Salmon head kidney -1 (SHK-1) cell lines were evaluated for isolation of SAV-1 from kidney samples of experimentally infected Atlantic salmon (Salmo salar). The presence of infection in these samples was confirmed both by cell culture and reverse transcription polymerase chain reaction (RT-PCR). Homogenates of kidney from fish 3 days post-infection (p.i.) were inoculated onto the three cell lines and the development of a cytopathic effect (CPE) recorded. The CHH-1 cells produced a rapid CPE from Day 6 p.i., while the CHSE-214 cells showed the presence of a CPE from Day 10 p.i. In comparison, a CPE developed much later in the SHK-1 cells, from Day 20 p.i. The virus was successfully isolated on all three cell lines in subsequent passages, indicating that CHSE-214, CHH-1, and SHK-1 cells can be used for the isolation and culture of SAV-1. The CHH-1 cell line, however, has proven the most useful, since the CPE developed the quickest in this cell line

An oral pH-responsive Streptococcus agalactiae vaccine formulation provides protective immunity to pathogen challenge in tilapia: A proof-of-concept study

Intensive tilapia farming has contributed significantly to food security as well as to the emergence of novel pathogens. This includes Streptococcus agalactiae or Group B Streptococcus (GBS) sequence type (ST) 283, which caused the first known outbreak of foodborne GBS illness in humans. An oral, easy-to-administer fish vaccine is needed to reduce losses in fish production and the risk of zoonotic transmission associated with GBS. We conducted a proof-of-concept study to develop an oral vaccine formulation that would only release its vaccine cargo at the site of action, i.e., in the fish gastrointestinal tract, and to evaluate whether it provided protection from experimental challenge with GBS. Formalin-inactivated S. agalactiae ST283, was entrapped within microparticles of Eudragit® E100 polymer using a double-emulsification solvent evaporation method. Exposure to an acidic medium simulating the environment in tilapia stomach showed that the size of the vaccine-loaded microparticles decreased rapidly, reflecting microparticle erosion and release of the vaccine cargo. In vivo studies in tilapia showed that oral administration of vaccine-loaded microparticles to fish provided significant protection from subsequent homologous pathogen challenge with GBS ST283 by immersion compared to the control groups which received blank microparticles or buffer, reducing mortality from 70% to 20%. The high efficacy shows the promise of the vaccine platform developed herein, which might be adapted for other bacterial pathogens and other fish species

Cellular and molecular pathogenesis of Salmonid alphavirus 1 in Atlantic salmon Salmo salar L

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 diseaseEThOS - Electronic Theses Online ServiceCommonwealth Scholarship CommissionGBUnited Kingdo

Pathogenesis of experimental salmonid alphavirus infection in vivo: an ultrastructural insight

Salmonid alphavirus (SAV) is an enveloped, single-stranded, positive sense RNA virus belonging to the familyTogaviridae. It causes economically devastating disease in cultured salmonids. The characteristic features of SAV infection include severe histopathological changes in the heart, pancreas and skeletal muscles of diseased fish. Although the presence of virus has been reported in a wider range of tissues, the mechanisms responsible for viral tissue tropism and for lesion development during the disease are not clearly described or understood. Previously, we have described membrane-dependent morphogenesis of SAV and associated apoptosis-mediated cell death in vitro. The aims of the present study were to explore ultrastructural changes associated with SAV infection in vivo. Cytolytic changes were observed in heart, but not in gill and head-kidney of virus-infected fish, although they still exhibited signs of SAV morphogenesis. Ultrastructural changes associated with virus replication were also noted in leukocytes in the head kidney of virus-infected fish. These results further describe the presence of degenerative lesions in the heart as expected, but not in the gills and in the kidney

Assessment of Marine Gill Disease in Farmed Atlantic Salmon (Salmo salar) in Chile Using a Novel Total Gross Gill Scoring System:A Case Study

Gill disorders have become more prevalent and widespread in finfish aquaculture in recent years. Their aetiology is often considered to be multifactorial. Effective diagnosis, control and prevention are hindered by the lack of standardised methodologies to characterise the aetiological agents, which produce an array of clinical and pathological presentations. The aim of this study was to define a novel gross pathological scoring system suitable for field-based macroscopic assessment of complex or multifactorial gill disease in farmed Atlantic salmon, using samples derived from a gill disease outbreak in Chile. Clinical assessment of gross gill morphology was performed, and gill samples were collected for qPCR and histology. A novel total gill scoring system was developed, which assesses gross pathological changes combining both the presumptive or healed amoebic gill disease (AGD) and the presence of other types of gill lesions. This scoring system offers a standardised approach to characterise the severe proliferative pathologies in affected gills. This total gill scoring system can substantially contribute to the development of robust mitigation strategies and could be used as an indicator trait for incorporating resistance to multifactorial gill disease into breeding goals

Interferon-mediated host response in experimentally induced salmonid alphavirus 1 infection in Atlantic salmon (Salmo salar L.)

Salmonid alphavirus (SAV) infection in cultured salmonids causes severe economic losses across Europe. Immune protection and antiviral mechanisms of the host against SAV are poorly characterised in vivo. Analysis of immune gene expression in head kidney of Atlantic salmon (Salmo salar L.) experimentally infected with SAV 1, using a quantitative reverse transcription polymerase chain reaction (qRT-PCR), revealed rapid induction of interferon I (INF-I), interferon II (INF-II) and INF-I associated Mx genes in SAV 1 infected fish compared to control fish injected with tissue culture supernatant. Mx protein was found to be highly expressed in the heart and mucosal membranes of infected fish by immunohistochemistry (IHC). Interestingly, the pathological changes that were observed in the target tissues of the virus became visible some time after peak expression of genes associated with the INF-I-pathway in head kidney tissue. These findings suggest that a non-specific antiviral immune response is rapidly induced during the early stages of SAV infection in salmon

Ulcerative dermatitis in wild dusky grouper Epinephelus marginatus (Lowe) from Libyan waters

In the period 2013–2015, wild dusky grouper,Epinephelus marginatus(Lowe), caught in Libyan coastal waters and ranging in size from 42 to 92cm in total length, were observed to have distinctive skin lesions of unknown aetiology. Histopathologically, the lesions comprised a multifocal, unilateral or bilateral dermatitis, involving the epidermis, superficial dermis and scale pockets, and sometimes, in severe cases, the hypodermis. Severe lesions had marked epidermal spongiosis progressing to ulceration. Healing was observed in some fish. Bacteria and fungi could be isolated from severe lesions, although they were not seen histopathologically in early-stage lesions. By contrast, metazoan parasite eggs were observed in the dermis and epidermis of some fish with mild and moderate dermatitis. Unidentified gravid digeneantrematode parasites carrying similar eggs were also seen within the blood vessels of the deep and superficial dermis. The cause of this distinctive condition, termed dusky grouper dermatitis (DGD), and its potential impact upon already threatened Mediterranean wild dusky grouper populations and upon cultured grouper more widely have yet to be established

Transcriptomic analysis of the host response to early stage salmonid alphavirus (SAV-1) infection in Atlantic salmon Salmo salar L.

Salmon pancreas disease, caused by salmonid alphavirus (SAV) of the family Togaviridae, is an economically important disease affecting farmed Atlantic salmon (Salmo salar L.) in Scotland, Norway, and Ireland. The virus causes characteristic lesions in the pancreas, heart, kidney and skeletal muscle of infected fish. The mechanisms responsible for the pathology and the immune responses elicited in infected Atlantic salmon are not fully understood. A microarray-based study was therefore performed to evaluate the host transcriptomic response during the early stages of an experimentally-induced SAV-1 infection. Atlantic salmon parr were injected intra-peritoneally with viral cell culture supernatant or cell culture supernatant without virus. RNA, extracted from head kidney sampled from infected and control fish at 1, 3 and 5 days post-injection (d.p.i.), was interrogated with the 17 k TRAITS/SGP cDNA microarray. The greatest number of significantly differentially expressed genes was recorded at 3 d.p.i., mainly associated with immune and defence mechanisms, including genes involved in interferon I pathways and Major Histocompatibility Complex Class I and II responses. Genes associated with apoptosis and cellular stress were also found to be differentially expressed between infected and uninfected individuals, as were genes involved in inhibiting viral attachment and replication. The microarray results were validated by follow-on analysis of eight genes by real-time PCR. The findings of the study reflect mechanisms used by the host to protect itself during the early stages of SAV-1 infection. In particular, there was evidence of rapid induction of interferon-mediated responses similar to those seen during mammalian alphavirus infections, and also early involvement of an adaptive immune response. This study provides essential knowledge to assist in the development of effective control and management strategies for SAV-1 infection

The effect of dietary n-3/n-6 polyunsaturated fatty acid ratio on salmonid alphavirus subtype 1 (SAV-1) replication in tissues of experimentally infected rainbow trout (Oncorhynchus mykiss)

Salmon pancreas disease (SPD) is one of the most commercially significant viral diseases of farmed Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) in Europe. In this study, the potential for dietary mitigation of the disease using different polyunsaturated fatty acid (PUFA) profiles was assessed in rainbow trout. We experimentally infected fish with salmonid alphavirus subtype 1 (SAV-1), the causative agent of SPD. These fish were fed two diets with different n-3/n-6 PUFA ratio (high omega 3, 3.08, and high omega 6, 0.87). We assessed the influence of the diets on the fatty acid composition of the heart at 0 days post infection (d.p.i.) (after 4 weeks of feeding the experimental diets prior to SAV-1 infection), and sampled infected and control fish at 5, 15 and 30d.p.i. Viral E1 and E2 glycoprotein genes were quantified by two absolute real-time PCRs in all the organs sampled, and significantly lower levels of the virus were evident in the organs of fish fed with high omega 6. Characteristic pathological lesions were identified in infected fish as early as 5d.p.i., with no significant differences in the pathology lesion scores between the two dietary regimes. This study shows that decreasing the n-3/n-6 PUFA ratio in experimental diets of rainbow trout changes the fatty acid content of the fish, and is associated with reduced SAV-1 replication in rainbow trout