Equine Interferon-Lambda

Rationale: Interferons (IFN) are a group of biological regulatory proteins, or cytokines, which act as an early line of defense against viral infections. The Type-1 interferon response (IFN-alpha/beta or á/â) is known to be important to innate immune responses by inducing a non-specific antiviral state, and now it is recognized as important for strong adaptive (humoral/CTL) immune responses to some viruses. But, we suggest that IFN-á/â are not the only antiviral IFNs involved in viral respiratory infections in the horse. In the horse, Type-III IFN or IFN-lambda (IFN-ë) has not been studied at all. This is an important gap in knowledge relevant to the American Quarter Horse Association’s mission of advancing the health and well-being of horses. In several other species, it is now known that IFN-ë is the predominant IFN type secreted by epithelial cells in response to virus infections such as influenza, and an important contributor to innate immunity. In the equine genome there are candidate sequences corresponding to the subclasses IFN-ë1, IFN-ë2, and IFN-ë3, which were identified using gene predictions from other species. But the functional annotation of these sequences has not been reported; that is, nothing is known about expression and function of IFN-ë in the horse. Our goal is to begin this analysis. Hypothesis/Objectives: Our hypothesis is that equine epithelial cells express cytokines of the IFN-ë family in response to stimulation with poly-I:C and to virus infection with influenza or equine herpesvirus-1 (EHV-1). Specific objectives include: 1). To identify, clone, and characterize equine IFN-ë gene products. 2) To evaluate IFN-ë expression in response to equine influenza (EIV) and EHV-1 infection in an equine epithelial cell culture model. Study Design: We propose to employ our expertise in genomics and viral immunology to create primers and probes for mRNAs of the different equine IFN-ë subclasses. We will use these (a) to clone and sequence the gene products, and (b) to quantitatively evaluate IFN-ë expression using our established model system of equine primary epithelial cell cultures infected with equine respiratory pathogens including EIV and EHV-1. Such cells are a useful model because they are susceptible to infection with EIV and EHV-1, and believed to be capable of expressing and responding to IFN-ë. No live animal work is required for this project. Preliminary Data: Using primers we have designed based on candidate sequences in the equine genome, we have already amplified PCR products of the correct size for each of the 3 candidate IFN-ë genes, from equine genomic DNA. Sequencing of the IFN-ë1 amplicon is complete, confirms its identity, and is being used to amplify the rest of the gene. Sequencing of the other PCR products is in progress. Expected Results: We expect to develop a set of tools for detection and quantitation of IFN-ë mRNA in cells of the horse. We expect to obtain plasmids containing the full-length coding sequences, which will confirm or correct the predicted exon structures and identify whether a subclass is a functional gene or a pseudogene. Based on reported results from other species, we expect to find that in response to one or both of the viruses, EIV and EHV-1, one or more subclasses of equine IFN-ë are secreted in epithelial cells at levels comparable to IFN-á. If we find that IFN-ë has been an unrecognized important player in equine innate immune responses, this project will open the door to future work to characterize its roles in detail. Potential Impact for the Horse Industry: this is the possibility that with understanding of the role of IFN-ë in equine respiratory viral infections, we can assess whether IFN-ë responses are markers of virus pathogenicity in the horse and whether these responses can be manipulated to reduce disease severity in the horse, i.e. design interventions to bolster innate or adaptive immune responses against EIV or particularly EHV-1, leading to improved control of these diseases. New approaches to control of EHV-1 are particularly needed. Based on findings in humans, IFN-ë may also prove eventually to play a role in other equine diseases of epithelial tissues, such as equine inflammatory airway disease. This information will be applicable to all breeds of horses.