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High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes
Published on: March 24, 2015
Expression and biological activity of two types of interferon genes in medaka (Oryzias latipes)
Shun Maekawa1, Yi-An Chiang1, Jun-ichi Hikima2
1Institute of Biotechnology, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan.
Abstract:
Type I interferon (IFN) is one of most important cytokines for antiviral responses in fish innate immunity, after the induction pathway following pattern recognition. In this study, 2 types of type I IFN mRNA from a medaka (Japanese rice fish; Oryzias latipes) were identified and classified (phylogenetic analysis) into subgroup-a and -d by (designated olIFNa and olIFNd, respectively). Both olIFNa and olIFNd (encoding 197 and 187 amino acid residues, respectively) contained 2 cysteines. Gene expression pattern of olIFNa, olIFNd and IFN-stimulated genes (ISGs) was assessed (quantitative real-time reverse transcriptase PCR, qRT-PCR) in various organs (i.e., whole kidney, liver and spleen) of medaka stimulated by polyI:C or infected with nervous necrosis virus (NNV). Expression of olIFNa, olIFNd and ISGs, especially the ISG15 gene, were significantly upregulated after NNV-infection. Furthermore, olIFNa, olIFNd and ISGs mRNAs were sufficiently induced in DIT cells (i.e., medaka hepatoma cell line) transfected with polyI:C or infected with NNV. In addition, in vitro biological activities of recombinant olIFNa and olIFNd (rolIFNa and rolIFNd) produced by mammalian cell line HEK293T were also characterized. Expression of GIG1a and ISG15 genes in kidney cells of adult medaka were induced by rolIFNa or rolIFNd. The olIFNs-overexpressing DIT cells had reduced viral titers following NNV infection. Therefore, we inferred that 2 type I IFNs were involved in innate immunity (antiviral response) in medaka fish.
Insights
Two type I interferons (IFNs) in medaka fish, olIFNa and olIFNd, were identified and shown to play a crucial role in antiviral innate immunity against nervous necrosis virus (NNV). Their expression is upregulated upon NNV infection, conferring protection.
Area of Science:
- * Molecular Biology
- * Immunology
- * Fish Aquaculture
Background:
- * Type I interferons (IFNs) are critical cytokines in antiviral innate immunity, particularly in fish.
- * Understanding fish IFN responses is vital for managing viral diseases in aquaculture.
- * Medaka fish (Oryzias latipes) serve as a valuable model organism for studying fish immunology.
Purpose of the Study:
- * To identify and characterize type I IFN genes in medaka fish.
- * To investigate the expression patterns and antiviral functions of these medaka IFNs (olIFNs) in response to viral infection.
- * To elucidate the role of olIFNs in the innate immune response of medaka.
Main Methods:
- * Phylogenetic analysis to classify two types of medaka type I IFN mRNA (olIFNa and olIFNd).
- * Quantitative real-time reverse transcriptase PCR (qRT-PCR) to assess gene expression in various organs and cell lines.
- * In vitro characterization of recombinant olIFNs (rolIFNs) biological activities and assessment of viral titers in olIFN-overexpressing cells.
Main Results:
- * Two medaka type I IFNs, olIFNa and olIFNd, were identified and phylogenetically classified.
- * Expression of olIFNa, olIFNd, and IFN-stimulated genes (ISGs), notably ISG15, was significantly upregulated in medaka organs and DIT cells upon infection with nervous necrosis virus (NNV) or stimulation with polyI:C.
- * Recombinant olIFNs induced ISG expression in medaka kidney cells, and olIFN-overexpressing cells exhibited reduced viral titers after NNV infection.
Conclusions:
- * Medaka fish possess at least two distinct type I IFNs, olIFNa and olIFNd, involved in antiviral defense.
- * These olIFNs are key components of the innate immune response in medaka, activated by viral infection.
- * The study provides insights into the molecular mechanisms of antiviral immunity in fish, relevant for disease control in aquaculture.
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