Cholesterol is essential for building and maintaining cell membranes and is critical for several steps in the replication cycle of viruses, especially for enveloped viruses. In mammalian cells virus infections lead to the accumulation of the oxysterol 25-hydroxycholesterol (25HC), an antiviral factor, which is produced from cholesterol by the cholesterol 25 hydroxylase (CH25H). Antiviral responses based on CH25H are not well studied in fish. Therefore, in the present study putative genes encoding for CH25H were identified and amplified in common carp and rainbow trout cells and an HPLC-MS method was applied for determination of oxysterol concentrations in these cells under virus infection. Our results give some evidence that the activation of CH25H could be a part of the antiviral response against a broad spectrum of viruses infecting fish, in both common carp and rainbow trout cells in vitro. Quantification of oxysterols showed that fibroblastic cells are capable of producing 25HC and its metabolite 7α,25diHC. The oxysterol 25HC showed an antiviral activity by blocking the entry of cyprinid herpesvirus 3 (CyHV-3) into KFC cells, but not spring viremia of carp virus (SVCV) or common carp paramyxovirus (Para) in the same cells, or viral haemorrhagic septicaemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) into RTG-2 cells. Despite the fact that the CH25H based antiviral response coincides with type I IFN responses, the stimulation of salmonid cells with recombinant type I IFN proteins from rainbow trout could not induce ch25h_b gene expression. This provided further evidence, that the CH25H-response is not type I IFN dependent. Interestingly, the susceptibility of CyHV-3 to 25HC is counteracted by a downregulation of the expression of the ch25h_b gene in carp fibroblasts during CyHV-3 infection. This shows a unique interplay between oxysterol based immune responses and immunomodulatory abilities of certain viruses.

PLAAT1 is a member of the PLAAT protein family and plays important roles in tumor suppression, transglutaminase activation and peroxisomal biogenesis. Recently, PLAAT1 has been shown to promote degradation of p53 protein and cellular organelles such as mitochondria, endoplasmic reticulum and lysosome. In this study, we show that PLAAT1 inhibits the production of type I interferon and promotes virus replication in zebrafish. Overexpression of Plaat1 in zebrafish cells suppresses antiviral responses and promotes virus replication. Mechanistically, PLAAT1 interacts with IRF3 and IRF7 to initiate degradation of IRF3 and IRF7, which can be attenuated by 3-methyladenine, an inhibitor of autophagosome. Our study provides novel insights into the functions of PLAAT1 in host immune response to viral infection.

Interferons orchestrate host antiviral responses in jawed vertebrates. They are categorized into three classes; IFN1 and IFN3 are the primary antiviral cytokine lineages, while IFN2 responds to a broader variety of pathogens. The evolutionary relationships within and between these three classes have proven difficult to resolve. Here, we reassess interferon evolution, considering key phylogenetic pitfalls including taxon sampling, alignment quality, model adequacy, and outgroup choice. We reveal that cartilaginous fishes, and hence the jawed vertebrate ancestor, possess(ed) orthologs of all three interferon classes. We show that IFN3 groups sister to IFN1, resolve the origins of the human IFN3 lineages, and find that intronless IFN3s emerged at least three times. IFN2 genes are highly conserved, except for IFN-γ-rel, which we confirm resulted from a teleost-specific duplication. Our analyses show that IFN1 phylogeny is highly sensitive to phylogenetic error. By accounting for this, we describe a new backbone IFN1 phylogeny that implies several IFN1 genes existed in the jawed vertebrate ancestor. One of these is represented by the intronless IFN1s of tetrapods, including mammalian-like repertoires of reptile IFN1s and a subset of amphibian IFN1s, in addition to newly-identified intron-containing shark IFN1 genes. IFN-f, previously only found in teleosts, likely represents another ancestral jawed vertebrate IFN1 family member, suggesting the current classification of fish IFN1s into two groups based on the number of cysteines may need revision. The providence of the remaining fish IFN1s and the coelacanth IFN1s proved difficult to resolve, but they may also be ancestral jawed vertebrate IFN1 lineages. Finally, a large group of amphibian-specific IFN1s falls sister to all other IFN1s and was likely also present in the jawed vertebrate ancestor. Our results verify that intronless IFN1s have evolved multiple times in amphibians and indicate that no one-to-one orthology exists between mammal and reptile IFN1s. Our data also imply that diversification of the multiple IFN1s present in the jawed vertebrate ancestor has occurred through a rapid birth-death process, consistent with functional maintenance over a 450-million-year host-pathogen arms race. In summary, this study reveals a new model of interferon evolution important to our understanding of jawed vertebrate antiviral immunity.

Teleost type I interferons (IFNs) are categorized into group I and II subgroups that bind to distinct receptors to activate antiviral responses. However, the interaction between ifn ligands and receptors has not fully been understood. In this study, the crystal structure of grass carp [Ctenopharyngodon idella (Ci)] IFNa has been solved at 1.58Å and consists of six helices. The CiIFNa displays a typical structure of type I IFNs with a straight helix F and lacks a helix element in the AB loop. Superposition modeling identified several key residues involved in the interaction with receptors. It was found that CiIFNa bound to cytokine receptor family B (CRFB) 1, CRFB2, and CRFB5, and the three receptors could form heterodimeric receptor complexes. Furthermore, mutation of Leu27, Glu103, Lys117, and His165 markedly decreased the phosphorylation of signal transducer and activator of transcription (STAT) 1a induced by CiIFNa in the Epithelioma papulosum cyprini (EPC) cells, and Glu103 was shown to be required for the CiIFNa-activated antiviral activity. Interestingly, wild-type and mutant CiIFNa proteins did not alter the phosphorylation levels of STAT1b. Our results demonstrate that fish type I IFNs, although structurally conserved, interact with the receptors in a manner that may differ from mammalian homologs.

Cartilaginous fishes are the most evolutionary-distant vertebrates from mammals and possess an immunoglobulin (Ig)- and T cell-mediated adaptive immunity. CD8 is the hallmark receptor of cytotoxic T cells and is required for the formation of T cell receptor-major histocompatibility complex (TCR-MHC) class I complexes.

Interleukin (IL)-22 plays an important role in regulating inflammation and clearance of infectious pathogens. IL-22 homologs have been discovered in fish, but the functions and sources of IL-22 have not been fully characterized. In this study, an IL-22 homolog was identified in grass carp and its bioactivities were investigated. The grass carp IL-22 was constitutively expressed in tissues, with the highest expression detected in the gills and hindgut. It was upregulated in the spleen after infection with Flavobacterium columnare and grass carp reovirus and in the primary head kidney and spleen leukocytes stimulated with LPS and IL-34. Conversely, it was downregulated by Th2 cytokines such as IL-4/13B and IL-10. The recombinant IL-22 produced in bacteria showed a stimulatory effect on the expression of inflammatory cytokines and STAT3 in the primary head kidney leukocytes and CIK cells. Moreover, the IL-22-positive cells were found to be induced in the hindgut and head kidney 24 h after infection by F. columnare. Our data suggest that IL-22 plays an important role in regulating mucosal and systemic immunity against bacterial and viral infection.