الفهرس 
AcknowledgmentOnly 14 pages are availabe for public view
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Abstract
Spring viremia of carp (SVC) is a contagious viral disease that necessitates early notification to the World Organization for Animal Health (Office International des Epizooties, OIE). SVC causes high mortality among infected fish so its outbreaks can cause significant economic losses. The immune responses to Spring viremia of carp virus and the exact mechanisms by which the virus induces pathogenesis are poorly understood. This study aimed to investigate the modulatory effects of SVCV infection on immune- related genes.
Nile tilapia were immersed in SVCV (3.2×107 TCID50/ml) for 4 hrs at 14±0.5°C. Nine fish (3 fish from each of triplicates) were randomly selected from different aquaria of the treated and control groups at different time points (1sth, 12th hrs., 1std, 3rd d, 5th d, 7th d, 11th d, 14th d and 21th d) post infection (pi). Fish were euthanized and gills, kidney, spleen, and intestine were sampled. Expression of the target genes (IL-1β, TNF-α, IL-10, IFN-γ, Mx andSOCS3) were estimated using the tilapia actin and elongation factor as internal controls.
In gills, IL-1β was upregulated by 1.8-fold at the 12th hpi When compared to the control. It was most expressed at the 3rd dpi, when it was upregulated by 4.3-fold. Upregulation was still present at the 5th and 7th dpi but at lower levels, recording 3 and 1.9-folds as the control, respectively. IL-10 was discovered to be 1.9-fold upregulated at the 12th hpi When compared to the control. It continued to be upregulated until reaching its peak of 4.9-fold at the 5th dpi, and it continued to be upregulated at the 7th and 11th dpi to record 3.5 and 1.5-folds as the control, respectively. IFN-γ elicited an expression of 1.5-fold as the control at 1st dpi. It was most expressed at the 3rd dpi, when it was upregulated by 3.4-fold. Upregulation was still present at the 5th and 7th dpi, but at lower levels, recording 2.6 and 1.5-folds as the control, respectively. The SOCS3 gene was significantly upregulated reaching 2.9-fold at the 3rd dpi in comparison to control. It was upregulated all the way up to the 5th dpi, when it peaked at 5.9-fold, and up to the 7th and 11th dpi but at lower levels, to record 3.5 and 1.5-folds as the control, respectively. Mx was upregulated by 1.8-fold at 1st dpi Comparatively to the control. and still upregulated until reach its highest expression of 4.3-fold at 3rd dpi and continued in its upregulation but at lesser levels at the 5th, 7th and 11th dpi recording 3.1, 2.2 and 1.5-folds as the control, respectively. Consequently, when compared the expression of all studied genes per time point, IL-1β and IL-10 were the two cytokines that were most noticeable in gills at the 12th hpi and the 1st dpi. IL-Iβ and Mx were considerably elevated in contrast to the other genes during the third day after infection. The top expressed cytokines at the 5th and 7th were IL-10 and SOCS3. At the 11th dpi, SOCS3 and Mx were considerably elevated in contrast to the other genes. Insignificant alterations were seen among all studied genes starting on day 14 post-infection.
In kidney, IL-1β was discovered to be upregulated at an early stage of sampling, recording 1.4-fold as the control at 12th hpi. It was upregulated all the way up to the 3rd dpi, when it peaked at 3.8-fold, and up to the 5th,7th and 11th dpi but at lower levels, to record 2.9, 1.9 and 1.3-folds as the control, respectively. IL-10 expressed 2.3, 2.6-folds higher than the control at 12th hpi and 1st dpi, respectively that were statistically insignificant. and still upregulated until reach its highest expression of 4.3-fold at 5th dpi and continued in its upregulation but at lesser levels at the 7th and 11th dpi recording 2.8 and 1.9-folds as control. INF-γ found to be elevated at an early stage of sampling, at the 12th hpi, it reached 1.5 -fold when compared to the control and showed the peak at the 3rd dpi, where it reached 3.8-fold and still upregulated but with less values at the 5th, and 7th dpi, recording 2.7 and 2.1-folds as that of control, respectively. The SOCS3 gene elicited an expression of 2.2-fold as the control at 1st dpi. It was most expressed at the 5th dpi, when it was upregulated by 5.2-fold. Then, the expression down-regulated reached 2.3-fold at 7th dpi Comparatively to the control. Mx was significantly upregulated in comparison with control at 12th hpi by 1.5-fold. Upregulation was still present at the 3rd dpi, when it peaked at 5.5-fold, and up to the 5th,7th and 11th dpi but at lower levels, to record 4.5, 3.4 and 2-folds as the control, respectively. Consequently, when compared the expression of all studied genes per time point, IL-10 was the most noticeable in the kidney at the 12th hpi. At the 1st dpi, IL-10, IFN-γ and SOCS3 were considerably the highest in contrast to the expressions of other genes that were statistically insignificant. On the other hand, Mx was considerably the highest in contrast to the expressions of other genes at 3rd d, 7th d and 11th dpi. The top expressed cytokine at the 5th dpi was SOCS3. Insignificant alterations were seen among all studied genes starting on day 14 post-infection.
In spleen, IL-1β was upregulated at the 12th hpi exhibiting 2.1-fold as that of control and reached its maximum expression with 3.4-fold as the control at 3rd dpi. Then, the expression down-regulated reached 2.1 and 1.3-fold higher than the control at 5th and 7th dpi, respectively. TNF-α found to be upregulated at an early stage of infection. At the 12th hpi, it reached 2.3-fold when compared to the control and showed the highest expression at the 3rd dpi, where it reached 6.1-fold and still upregulated but with less values at the 5th and 7th dpi, recording 3.9 and 2.5-folds as that of control, respectively. IL-10 expressed 1.4, 1.5-folds higher than the control at 12th hpi and 1st dpi, respectively that were statistically insignificant. and still upregulated until reach its highest expression of 4.4-fold at 5th dpi and continued in its upregulation but at lesser levels at the 7th dpi recording 2.1-fold as control. IFN-γ was discovered to be 1.7-fold upregulated at the 12th hpi When compared to the control. It continued to be upregulated until reaching its peak of 3.7-fold at the 3rd dpi, and it continued to be upregulated at the 5th dpi to record 1.7-fold as the control. The SOCS3 gene elicited an expression of 1.4-fold as the control at 1st dpi. It was most expressed at the 5th dpi, when it was upregulated by 5.1-fold as the control. Upregulation was still present at the 7th dpi, but at lower levels, recording 2.7-fold as the control. Mx discovered to be upregulated at an early stage of sampling, recording 1.6-fold as the control at 12th hpi. It was upregulated all the way up to the 3rd dpi, when it reached its maximum expression with 5.4-fold, and up to the 5th and 7th dpi but at lower levels, to record 2.8 and 2.2-folds as the control, respectively. Consequently, when compared the expression of all studied genes per time point, IL-1β and TNF-α were the most prominent cytokines in the spleen at the 12th hpi. At the 1st d and 3rd dpi, TNF-α was considerably the highest in contrast to the expressions of other genes. The top expressed cytokine at the 5th d and 7th d post-infection was SOCS3. Insignificant alterations were seen among all studied genes starting on day 11 post-infection.
In intestine, IL-1β was upregulated by 1.4-fold at the 12th hpi When compared to the control. It was most expressed at the 3rd dpi, when it was upregulated by 3.6-fold. Upregulation was still present at the 5th and 7th dpi but at lower levels, recording 2.4 and 1.8-folds as the control, respectively. IL-10 was discovered to be 2-fold upregulated at the 12th hpi When compared to the control. It continued to be upregulated until reaching its peak of 4.8-fold at the 5th dpi, and it continued to be upregulated at the 7th and 11th dpi to record 2.7 and 1.5-folds as the control, respectively. INF-γ found to be elevated at an early stage of sampling, at the 12th hpi, it reached 1.5 -fold when compared to the control and showed the peak at the 3rd dpi, where it reached 3.2-fold and still upregulated but with less values at the 5th, and 7th dpi, recording 2.4 and 1.6-folds as that of control, respectively. The SOCS3 gene was discovered to be upregulated at an early stage of sampling, recording 2.2-fold as the control at 12th hpi. It was upregulated all the way up to the 5th dpi, when it reached the highest expression at 5.5-fold, and up to the 7th and 11th dpi but at lower levels, to record 4.2 and 2.2-folds as the control, respectively. Mx was significantly upregulated in comparison with control at 12th hpi by 2.3-fold. Upregulation was still present at the 3rd dpi, when it reached maximum fold at 4.7-fold, and at the 5th,7th and 11th dpi but at lower levels, to record 3.6, 2.9 and 2.1-folds as the control, respectively. Consequently, when compared the expression of all studied genes per time point, IL-10, SOCS3, and Mx were the most pronounced gene expressed in the intestine at 12th hpi. At the 1st d and 3rd dpi Mx was considerably the highest in contrast to the expressions of other genes. SOCS3 and IL-10 were the most significant cytokines expressed at the 5th dpi. SOCS3 was considerably the highest in contrast to the expressions of other genes at the 7th dpi. SOCS3 and Mx were the most significant cytokines expressed at the 11th dpi. Insignificant alterations were seen among all studied genes starting on day 14 post-infection.
The IL-1β gene was expressed in all examined organs with the highest gene expression at 3rd dpi and have been detected in the gills followed by kidney, spleen and intestine. IL-10 and SOCS3 genes were expressed in all examined organs with the highest gene expression at 5th dpi and have been detected in the gills and intestine followed by kidney and spleen. INF-γ and Mx genes were expressed in all examined organs with the highest gene expression at 3rd dpi and have been detected in the kidney and spleen followed by gills and intestine.
When comparing the fold changes of each gene in all examined organs per each time point, the fold changes of IL-1β were noticeably highest in the gills and spleen at the (12th h and 1st d) post-infection, in the gills at 3rd dpi, in the gills and kidney at the (5th d and 7th d) post-infection and Insignificant alterations were seen among all studied organs starting from day 11 post-infection. the fold changes of IL-10 were noticeably highest in the kidney and intestine at the (12th h and 1st d) post-infection, in the spleen and intestine at 3rd dpi, in the gills and intestine at the 5th dpi, in the gills at 7th dpi, in the kidney at 11th dpi and Insignificant alterations were seen among all studied organs starting from day 14 post-infection. the fold changes of SOCS3 were noticeably highest in the intestine at the (12th h and 1st d) post-infection, in the spleen, kidney and intestine at 3rd dpi, in the gills and intestine at the 5th dpi, in the intestine at the (7th d and 11th d) post-infection and Insignificant alterations were seen among all studied organs starting from day 14 post-infection. the fold changes of IFN-γ were noticeably highest in the spleen, kidney and intestine at the 12th hpi, in the spleen and kidney at the (1st d and 3rd d) post-infection, in the gills and kidney at the 5th dpi, in the kidney at 7th dpi and Insignificant alterations were seen among all studied organs starting from day 11 post-infection. the fold changes of Mx were noticeably highest in the intestine at the 12th hpi, in the spleen and intestine at the 1st dpi, in the spleen and kidney at the 3rd dpi, in the kidney at the 5th dpi, in the kidney and intestine at the (7th d and 11th d) post-infection and Insignificant alterations were seen among all studied organs starting from day 14 post-infection.
Heatmap was performed using the expression function within heatmapper. column expression values are represented as color scale from red for lower expressions to black for higher expressions, it showed the black color of the highest expression of IL-1β, IFN-γ and Mx at the third day post-infection while, the black color of the highest expression of IL-10 and SOCS3 at the 5th day post-infection.
The heatmap with dendrogram of row clustering was used to demonstrate the similarity and differences in co-expression between genes across different time points, it showed that IL-1β, IFN-γ and Mx were clustered together, and IL-10 and SOCS3 were clustered together.