CD4+ T lymphocyte responses to viruses and virus-relevant stimuli in teleost fish.

Fish diseases caused by viruses are a major threat to aquaculture. Development of disease protection strategies for sustainable fish aquaculture requires a better understanding of the immune mechanisms involved in antiviral defence. The innate and adaptive arms of the vertebrate immune system collaborate to mount an effective defence against viral pathogens. The T lymphocyte components of the adaptive immune system, comprising two major classes (helper T, Th or CD4+ and cytotoxic T lymphocytes, CTLs or CD8+ T cells), are responsible for cell-mediated immune responses. In particular, CD4+ T cells and their different subsets orchestrate the actions of various other immune cells during immune responses, making CD4+ T cells central drivers of responses to pathogens and vaccines. CD4+ T cells are also present in teleost fish. Here we review the literature that reported the use of antibodies against CD4 in a few teleost fish species and transcription profiling of Th cell-relevant genes in the context of viral infections and virus-relevant immunomodulation. Studies reveal massive CD4+ T cell proliferation and expression of key cytokines, transcription factors, and effector molecules that evoke mammalian Th cell responses. We also discuss gaps in the current understanding and evaluation of teleost CD4+ T cell responses and how development and application of novel tools and approaches to interrogate such responses could bridge these gaps. A greater understanding of fish Th cell responses will further illuminate the evolution of vertebrate adaptive immunity, inform strategies to address viral infections in aquaculture, and could further foster fish as model organisms.

Infection and microbial molecular motifs modulate transcription of the interferon-inducible gene ifit5 in a teleost fish.

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are involved in antiviral defense. Members of this protein family contain distinctive multiple structural motifs comprising tetratricopeptides that are tandemly arrayed or dispersed along the polypeptide. IFIT-encoding genes are upregulated by type I interferons (IFNs) and other stimuli. IFIT proteins inhibit virus replication by binding to and regulating the functions of cellular and viral RNA and proteins. In teleost fish, knowledge about genes and functions of IFITs is currently limited. In the present work, we describe an IFIT5 orthologue in Atlantic salmon (SsaIFIT5) with characteristic tetratricopeptide repeat motifs. We show here that the gene encoding SsaIFIT5 (SsaIfit5) was ubiquitously expressed in various salmon tissues, while bacterial and viral challenge of live fish and in vitro stimulation of cells with recombinant IFNs and pathogen mimics triggered its transcription. The profound expression in response to various immune stimulation could be ascribed to the identified IFN response elements and binding sites for various immune-relevant transcription factors in the putative promoter of the SsaIfit5 gene. Our results establish SsaIfit5 as an IFN-stimulated gene in A. salmon and strongly suggest a phylogenetically conserved role of the IFIT5 protein in antimicrobial responses in vertebrates.

Two Virus-Induced MicroRNAs Known Only from Teleost Fishes Are Orthologues of MicroRNAs Involved in Cell Cycle Control in Humans.

MicroRNAs (miRNAs) are ~22 base pair-long non-coding RNAs which regulate gene expression in the cytoplasm of eukaryotic cells by binding to specific target regions in mRNAs to mediate transcriptional blocking or mRNA cleavage. Through their fundamental roles in cellular pathways, gene regulation mediated by miRNAs has been shown to be involved in almost all biological phenomena, including development, metabolism, cell cycle, tumor formation, and host-pathogen interactions. To address the latter in a primitive vertebrate host, we here used an array platform to analyze the miRNA response in rainbow trout (Oncorhynchus mykiss) following inoculation with the virulent fish rhabdovirus Viral hemorrhagic septicaemia virus. Two clustered miRNAs, miR-462 and miR-731 (herein referred to as miR-462 cluster), described only in teleost fishes, were found to be strongly upregulated, indicating their involvement in fish-virus interactions. We searched for homologues of the two teleost miRNAs in other vertebrate species and investigated whether findings related to ours have been reported for these homologues. Gene synteny analysis along with gene sequence conservation suggested that the teleost fish miR-462 and miR-731 had evolved from the ancestral miR-191 and miR-425 (herein called miR-191 cluster), respectively. Whereas the miR-462 cluster locus is found between two protein-coding genes (intergenic) in teleost fish genomes, the miR-191 cluster locus is found within an intron of a protein-coding gene (intragenic) in the human genome. Interferon (IFN)-inducible and immune-related promoter elements found upstream of the teleost miR-462 cluster locus suggested roles in immune responses to viral pathogens in fish, while in humans, the miR-191 cluster functionally associated with cell cycle regulation. Stimulation of fish cell cultures with the IFN inducer poly I:C accordingly upregulated the expression of miR-462 and miR-731, while no stimulatory effect on miR-191 and miR-425 expression was observed in human cell lines. Despite high sequence conservation, evolution has thus resulted in different regulation and presumably also different functional roles of these orthologous miRNA clusters in different vertebrate lineages.

Involvement of two microRNAs in the early immune response to DNA vaccination against a fish rhabdovirus.

Mechanisms that account for the high protective efficacy in teleost fish of a DNA vaccine expressing the glycoprotein (G) of Viral hemorrhagic septicemia virus (VHSV) are thought to involve early innate immune responses mediated by interferons (IFNs). Microribonucleic acids (miRNAs) are a diverse class of small (18-22 nucleotides) endogenous RNAs that potently mediate post-transcriptional silencing of a wide range of genes and are emerging as critical regulators of cellular processes, including immune responses. We have recently reported that miR-462 and miR-731 were strongly induced in rainbow trout infected with VHSV. In this study, we analyzed the expression of these miRNAs in fish following administration of the DNA vaccine and their potential functions. Quantitative RT-PCR analysis revealed the increased levels of miR-462, and miR-731 in the skeletal muscle tissue at the site of vaccine administration and in the liver of vaccinated fish relative to empty plasmid backbone-injected controls. The increased expression of these miRNAs in the skeletal muscle correlated with the increased levels of the type I interferon (IFN)-inducible gene Mx, type I IFN and IFN-γ genes at the vaccination site. Intramuscular injection of fish with either type I IFN or IFN-γ plasmid construct resulted in the upregulation of miR-462 and miR-731 at the site of injection, suggesting that the induction of these miRNAs is elicited by IFNs. To analyze the function of miR-462 and miR-731, specific silencing of these miRNAs using anti-miRNA oligonucleotides was conducted in poly I:C-treated rainbow trout fingerlings. Following VHSV challenge, anti-miRNA-injected fish had faster development of disease and higher mortalities than control fish, indicating that miR-462/731 may be involved in IFN-mediated protection conferred by poly I:C.