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Krishnan, Rahul,Qadiri, Syed Shariq Nazir,Kim, Jong-Oh,Kim, Jae-Ok,Oh, Myung-Joo The Korean Society of Fisheries and Aquatic Scienc 2019 Fisheries and Aquatic Sciences Vol.22 No.12
Background: In the present study, we evaluated four commonly used housekeeping genes, viz., actin-β, elongation factor-1α (EF1α), acidic ribosomal protein (ARP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal references for quantitative analysis of immune genes in nervous necrosis virus (NNV)-infected seven-band grouper, Hyporthodus septemfasciatus. Methods: Expression profiles of the four genes were estimated in 12 tissues of healthy and infected seven-band grouper. Expression stability of the genes was calculated using the delta Ct method, BestKeeper, NormFinder, and geNorm algorithms. Consensus ranking was performed using RefFinder, and statistical analysis was done using GraphpadPrism 5.0. Results: Tissue-specific variations were observed in the four tested housekeeping genes of healthy and NNV-infected seven-band grouper. Fold change calculation for interferon-1 and Mx expression using the four housekeeping genes as internal references presented varied profiles for each tissue. EF1α and actin-β was the most stable expressed gene in tissues of healthy and NNV-infected seven-band grouper, respectively. Consensus ranking using RefFinder suggested EF1α as the least variable and highly stable gene in the healthy and infected animals. Conclusions: These results suggest that EF1α can be a fairly better internal reference in comparison to other tested genes in this study during the NNV infection process. This forms the pilot study on the validation of reference genes in Hyporthodus septemfasciatus, in the context of NNV infection.
Rahul Krishnan,Syed Shariq Nazir Qadiri,김종오,Jae-Ok Kim,오명주 한국수산과학회 2019 Fisheries and Aquatic Sciences Vol.22 No.4
Background: In the present study, we evaluated four commonly used housekeeping genes, viz., actin-β, elongation factor-1α (EF1α), acidic ribosomal protein (ARP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal references for quantitative analysis of immune genes in nervous necrosis virus (NNV)-infected seven-band grouper, Hyporthodus septemfasciatus. Methods: Expression profiles of the four genes were estimated in 12 tissues of healthy and infected seven-band grouper. Expression stability of the genes was calculated using the delta Ct method, BestKeeper, NormFinder, and geNorm algorithms. Consensus ranking was performed using RefFinder, and statistical analysis was done using GraphpadPrism 5.0. Results: Tissue-specific variations were observed in the four tested housekeeping genes of healthy and NNVinfected seven-band grouper. Fold change calculation for interferon-1 and Mx expression using the four housekeeping genes as internal references presented varied profiles for each tissue. EF1α and actin-β was the most stable expressed gene in tissues of healthy and NNV-infected seven-band grouper, respectively. Consensus ranking using RefFinder suggested EF1α as the least variable and highly stable gene in the healthy and infected animals. Conclusions: These results suggest that EF1α can be a fairly better internal reference in comparison to other tested genes in this study during the NNV infection process. This forms the pilot study on the validation of reference genes in Hyporthodus septemfasciatus, in the context of NNV infection.
Krishnan, Rahul,Kim, Jong-Oh,Kim, Jae-Ok,Qadiri, Syed Shariq Nazir,Kim, Soo-Jin,Oh, Myung-Joo Elsevier 2019 Aquaculture Vol.506 No.-
<P><B>Abstract</B></P> <P>Here, we report the characterization of four calcium-independent cell adhesion molecules, viz- <I>nectin-1, 2, 3</I> and <I>4</I> from seven-band grouper <I>Hyporthodus septemfasciatus</I> a high valued marine finfish susceptible for nervous necrosis virus (NNV) infection. Structural analysis of the nectin molecules revealed an N-terminal V domain, two constant C2 domains, a transmembrane domain, and a cytoplasmic tail. All the nectin molecules except <I>nectin-4</I> shared a C-terminal interaction motif (E/AxYV). Phylogenetic analysis revealed a close relationship between seven-band grouper nectins and zebrafish nectins. Also, nectins ubiquitously expressed in all the tissues of healthy fish with higher transcript abundance in the brain, gill and anterior kidney. Significant up-regulation of <I>nectin-4</I> expression was observed in the brain of NNV challenged fish followed by the gill and kidney. Further, docking simulations of nectins with NNV capsid proteins revealed a strong interaction with nectin-1 and nectin-4. Our findings suggest that nectins might have a possible role to play in NNV neuropathogenesis. The present observations provide potential clues for further elucidating the involvement of nectins in NNV pathogenesis and could help in the development of new antiviral therapeutics inhibiting the NNV entry to the cell.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Four nectin genes were identified and characterized from sevenband grouper, <I>Hyporthodus septemfasciatus.</I> </LI> <LI> The molecules belonged to immunoglobulin super family with distinct V-domain, two C2 domain, a transmembrane helix and C-terminal interaction motif </LI> <LI> Highest temporal expression and fold change after NNV challenge was observed for nectin-4 in brain </LI> <LI> Docking simulations demonstrated significant positive interaction of NNV coat proteins with grouper nectins. </LI> </UL> </P>