RESUMO
Despite increased global interest in Chinook salmon aquaculture, little is known of their viral immune defenses. This study describes the establishment and characterization of a continuous cell line derived from Chinook salmon spleen, CHSS, and its use in innate immune studies. Optimal growth was seen at 14-18⯰C when grown in Leibovitz's L-15 media with 20% fetal bovine serum. DNA analyses confirmed that CHSS was Chinook salmon and genetically different from the only other available Chinook salmon cell line, CHSE-214. Unlike CHSE-214, CHSS could bind extracellular dsRNA, resulting in the rapid and robust expression of antiviral genes. Receptor/ligand blocking assays confirmed that class A scavenger receptors (SR-A) facilitated dsRNA binding and subsequent gene expression. Although both cell lines expressed three SR-A genes: SCARA3, SCARA4, and SCARA5, only CHSS appeared to have functional cell-surface SR-As for dsRNA. Collectively, CHSS is an excellent cell model to study dsRNA-mediated innate immunity in Chinook salmon.
Assuntos
Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Interferon Tipo I/genética , Interferon Tipo I/imunologia , RNA de Cadeia Dupla/imunologia , Salmão/genética , Salmão/imunologia , Receptores Depuradores Classe A/genética , Receptores Depuradores Classe A/imunologia , Animais , Antivirais/imunologia , Linhagem Celular , Proteínas de Peixes/biossíntese , Pesqueiros , Expressão Gênica , Imunidade Inata/genética , Interferon Tipo I/biossíntese , Ploidias , RNA de Cadeia Dupla/genética , RNA Viral/genética , RNA Viral/imunologia , Salmão/virologia , Receptores Depuradores Classe A/biossíntese , Baço/imunologia , Baço/virologiaRESUMO
Class A scavenger receptors (SR-As) are a family of five surface receptors whose functions in mammals are associated with innate immunity; however, their role in fish immunity requires further elucidation. The present study identifies, performs sequence analysis, and constitutive transcript expression analysis for three SR-A family members, SCARA3, SCARA4 and SCARA5, from rainbow trout. This work will provide a basis for future studies on SR-A function and their role in innate immunity in this economically important fish.
Assuntos
Imunidade Inata/genética , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/imunologia , Receptores Depuradores Classe A/genética , Receptores Depuradores Classe A/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Filogenia , Receptores Depuradores Classe A/química , Alinhamento de Sequência/veterináriaRESUMO
Double-stranded RNA (dsRNA) is produced by almost all viruses during their replicative cycle and is a potent inducer of the innate antiviral immune response including inducing expression of type I interferons (IFNs) and interferon-stimulated genes (ISGs). During lytic virus infections intracellular dsRNA can escape into the extracellular space, where surface pattern recognition receptors, such as class A scavenger receptors (SR-As) facilitate its binding and entry into neighbouring cells. Studying extracellular dsRNA entry is difficult due to the ubiquitous expression profile and compensatory dsRNA binding characteristics of SR-As; a SR-A deficient cell line has yet to be identified. The present study suggests the Chinook salmon embryonic cell line, CHSE-214, as a model for studying extracellular dsRNA sensing in vitro. CHSE-214 is unable to bind and respond to extracellular dsRNA, can only respond to dsRNA when it is transfected into the cells, and is able to bind dsRNA when overexpressing human SR-AI. The applications for this model could include elucidating: dsRNA binding and entry mechanisms, including sequence and length effects, as well as SR-A and other putative surface dsRNA receptor ligand binding studies.
Assuntos
RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Salmão , Fatores de Processamento de Serina-Arginina/metabolismo , Animais , Linhagem Celular , Embrião não Mamífero , Ligação ViralRESUMO
Aequorin, the bioluminescent protein found in the jellyfish Aequorea sp., has been immobilized in a porous sol-gel glass environment. The luminescence from this protein is specifically triggered by the presence of calcium ions, thus offering exciting possibilities for the development of an optical biosensor for this cationic species. The luminescence emission spectrum has been measured from the aequorin protein after interaction with calcium ions. The intensity of the luminescence, measured at the peak maximum of 470 nm, for the encapsulated protein has been calibrated against calcium ion concentration. The characterization of the protein within the sol-gel matrix has been reported together with biosensing experiments using human sera and milk samples. The results suggest that the sol-gel encapsulated aequorin protein offers potential as a one shot bioluminescence based biosensor for the determination of calcium ions in such complex matrices.
