ABSTRACT
Domestic cats are commonly affected by viral pathogens that induce lengthy infections with fatal outcomes. Prevention of viral propagation is of primordial importance in shelters and catteries, where cats from different backgrounds have narrow contacts. Oligonucleotides (ODN) containing cytosine-phosphate-guanosine motifs of class A (CpG-A) are highly potent synthetic inducers of innate antiviral mechanisms. The aim of this study was to test their ability to modulate innate immune responses and prevent viral replication as stand-alone agents in the domestic cat. CpG-A stimulation of feline peripheral blood mononuclear cells (PBMCs) enhanced their proliferation, increased the presence of co-stimulatory molecules on their surface and influenced their gene expression profiles in an antiviral orientation. Incubation of the supernatants of CpG-A stimulated PBMCs with feline cell lines of epithelial and fibroblastic origin induced expression of the antiviral myxovirus resistance (Mx) gene in these target cells, which also showed enhanced resistance to feline viruses from five distinct families, namely Coronaviridae, Herpesviridae, Caliciviridae, Parvoviridae, and Retroviridae. Most importantly, subcutaneous administration of CpG-A in domestic cats systemically increased the expression of Mx, reaching maximal levels within 24 h. Plasma from treated cats could furthermore inhibit viral replication in vitro. Altogether, our data highlight the promising potential of CpG-A to induce a preventive antiviral state in the cat and to protect feline populations against a broad range of virus infections.
Subject(s)
Cat Diseases/immunology , Cat Diseases/prevention & control , Immunity, Innate , Oligodeoxyribonucleotides/pharmacology , Virus Diseases/veterinary , Viruses/classification , Viruses/immunology , Animals , Cat Diseases/virology , Cats , Cell Line , Cell Proliferation , Flow Cytometry/veterinary , Gene Expression Regulation , Leukocytes, Mononuclear/immunology , Male , Myxovirus Resistance Proteins/genetics , Myxovirus Resistance Proteins/metabolism , Oligodeoxyribonucleotides/administration & dosage , Random Allocation , Real-Time Polymerase Chain Reaction/veterinary , Species Specificity , Toll-Like Receptor 9/agonists , Virus Diseases/immunology , Virus Diseases/prevention & control , Virus Diseases/virology , Virus ReplicationABSTRACT
The innate immune system plays a central role in host defence against viruses. While many studies portray mechanisms in early antiviral immune responses of humans and mice, much remains to be discovered about these mechanisms in the cat. With the objective of shedding light on early host-virus interactions in felids, we have developed 12 real-time TaqMan(®) qPCR systems for feline genes relevant to innate responses to viral infection, including those encoding for various IFNα and IFNω subtypes, IFNß, intracellular antiviral factor Mx, NK cell stimulator IL-15 and effectors perforin and granzyme B, as well as Toll-like receptors (TLRs) 3 and 8. Using these newly developed assays and others previously described, we measured the relative expression of selected markers at early time points after viral infection in vitro and in vivo. Feline embryonic fibroblasts (FEA) inoculated with feline leukemia virus (FeLV) indicated peak levels of IFNα, IFNß and Mx expression already 6h after infection. In contrast, Crandell-Rees feline kidney (CrFK) cells inoculated with feline herpes virus (FHV) responded to infection with high levels of IFNα and IFNß only after 24h, and no induction of Mx could be detected. In feline PBMCs challenged in vitro with feline immunodeficiency virus (FIV), maximal expression levels of IFNα, ß and ω subtype genes as well as IL-15 and TLRs 3, 7 and 8 were measured between 12 and 24h after infection, whereas expression levels of proinflammatory cytokine gene IL-6 were consistently downregulated until 48h post inoculation. A marginal upregulation of granzyme B was also observed within 3h after infection. In an in vivo experiment, cats challenged with FIV exhibited a 2.4-fold increase in IFNα expression in blood 1 week post infection. We furthermore demonstrate the possibility of stimulating feline immune cells in vitro with various immune response modifiers (IRMs) already known for their immunostimulatory properties in mice and humans, namely Poly IC, Resiquimod (R-848) and dSLIM™, a synthetic oligonucleotide containing several unmethylated CpG motifs. Stimulation of feline PBMCs with dSLIM™ and R-848 effectively enhanced expression of IFNα within 12h by factors of 6 and 12, respectively, and Poly IC induced an increase in Mx mRNA expression of 28-fold. Altogether, we describe new molecular tools and their successful use for the characterization of innate immune responses against viruses in the cat and provide evidence that feline cells can be stimulated by synthetic molecules to enhance their antiviral defence mechanisms.
