Interferon-stimulated genes as resilience factors for PRRSV infection

Porcine Reproductive and Respiratory Syndrome (PRRS) is a panzootic viral disease of pigs that causes major economic losses. The causative agent of PRRS, PRRS virus (PRRSV) is a rapidly evolving small enveloped RNA virus. Whilst improvements have been effected with changes in husbandry and vaccination, PRRS still has major impacts on pig health and welfare. PRRS accounts for about a third of the cost of infectious disease to the US pig industry amounting to $650M per year. PRRS is the most costly disease to pig industries of Europe, China and North America and new PRRSV variants have the potential to be even more devastating as demonstrated by recent outbreaks of highly pathogenic variants of the virus in China and Southeast Asia. The presence of endemic PRRSV also supresses immunity and increases susceptibility to other infections, leading to complex disease and increased use of antimicrobials. PRRSV has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. Like all viruses PRRSV relies on the host cell machinery to replicate itself and generate new viral particles.

There is growing evidence from in vitro and in vivo challenge experiments and field studies that there is host genetic variation in responses to and outcomes of PRRSV infection. In particular genes involved in the cellular immune response, the interferon (IFN) response, have been found to be candidates for genetic resilience. However, a general issue with animal challenge experiments and even field studies is that they usually have insufficient power to dissect genetic effects as there are too few animals, which makes them miss resilience variants.

IFNs are a crucial first line of defence against viral infection, acting by triggering the expression and activation of a suite of antiviral proteins. They shape the adaptive immune response by triggering release of cytokines and chemokines. The IFN response is fundamentally involved in the host response to virus infections. Developing a better understanding of the IFN response and how viruses counteract its effects has important implications for how we treat viral infections and the development of vaccines. Type I IFNs are the main cytokines for innate immunity against viral infections and include various subtypes depending on the animal species. IFN-gamma is the sole representative of type II IFN. While type I IFNs are produced in most cell types in response to different viruses, type II IFN is produced by limited types of cells including natural killer cells, activated T lymphocytes, macrophages, and neurons.

While the antiviral nature of IFN has long been documented, the specific mechanisms of how IFN inhibits different viruses is poorly understood. Recently, more than 500 human genes induced by IFN have been individually cloned, allowing overexpression and investigation of their effects on viruses. This has proven to be a particularly powerful approach, resulting in significant discoveries on the action of IFN in human cells. In the BBSRC grant propoasal "Identification of interferon stimulated genes that restrict cross-species transmission of influenza A virus" (BB/S00114X/1) we generated an arrayed type I ISG library for the pig. In this proposal we will combine this unique tool with cutting-edge pluripotent stem cell technology and a newly generated pig type II ISG library to identify genes playing a role in restricting PRRSV infection. These genes will be further investigated to assess whether they are suitable candidates for genetic selection or genome editing in pigs or may be targeted by antiviral drugs to combat PRRSV infection.

This proposal will not only identify valid PRRS resilience genes or antiviral strategy but also generate valuable technical resources and protocols that can be used for other porcine pathogens, in particular ones infecting immune cells, such as African swine fever virus, salmonella typhimurium, or toxoplasma gondii.

Porcine Reproductive and Respiratory Syndrome (PRRS) is a panzootic viral disease of pigs that causes major economic losses. The causative agent of PRRS, PRRS virus (PRRSV) is a rapidly evolving small enveloped RNA virus infecting cells of the monocyte/macrophage lineage. There is growing evidence that genetic variation in the cellular immune response, the interferon (IFN) response, has an impact on resilience of pigs towards PRRSV infection.

Systematic analysis using arrayed Interferon Stimulated Gene (ISG) expression libraries have proven a powerful method for identifying key components of the IFN response in human cells. Here we will combine the unique tool of a type I porcine ISG library with cutting-edge pluripotent stem cell technology and a newly generated pig type II ISG library to identify genes playing a role in restricting PRRSV infection. The arrayed ISG libraries rely on lentiviral vectors encoding an ISG together with Red Fluorescent Protein (RFP).Transduced, ISG-expressing, cells fluoresce red. Cells are then infected, in our case, with PRRSV and stained for the nucleocapsid protein (N). Viral antigen expression in RFP-positive (ISG-expressing) cells is subsequently monitored by flow cytometry. ISGs that confer antiviral effects result in significant reductions in N staining among RFP-positive populations. Since PRRSV infects macrophages and there is currently no available porcine macrophage cell line, we will make use of porcine embryonic stem cell-derived macrophages for the screening with the ISG libraries.

The identified ISG candidate genes with resilience potential against PRRSV will be validated and their genetic variability and effects thereof on PRRSV infection investigated. We will compare these results with the outcome of previous experimental infections to identify potential genetic causality towards the infection outcome. Furthermore, we will investigate whether ISGs may be used as antiviral drug targets against PRRSV infection.

Who will benefit from this research?

The potential non-academic beneficiaries of this research include pig breeding companies, pig producers and ultimately the entire chain of users of pig products, including meat packers, processors, retailers and consumers. There are also potential benefits to the biotechnology sector.

How will they benefit from this research?

PRRS is the most costly disease to pig industries of Europe, China and North America and new PRRSV variants have the potential to be even more devastating. The presence of endemic PRRSV also supresses immunity and increases susceptibility to other infections, leading to complex disease and increased use of antimicrobials. Thus, the development of novel and/or more effective strategies to control PRRS will improve the sustainability of the pig industry and potentially reduce antimicrobial use and the cost of pig products.

In the pig breeding sector the research outputs will have the potential to inform future breeding programmes. The pig breeding industry has already incorporated selection for desirable disease resistance genes into breeding programmes. To date selection for disease resistance has been limited to diseases for which susceptibility is determined by a single major gene. Moreover, breeding for disease resistance is constrained by the nature of any genetic variation in susceptibility to infection. Whilst evidence for genetic variation in host responses to infection with PRRSV exists, the genetic control of these responses is polygenic and there is no evidence to date of major genes conferring complete resistance to PRRSV. The results from this study can directly impact the selection of additional loci involved in PRRSV resilience. With increasing capabilities to genetically modify farmed animals there are also opportunities to engineer resistance. The recent demonstration that such genetically-engineered, PRRSV-resistant pigs can be generated shows the potential impact of such pigs for the pig industry. However, safety concerns over the highly mutagenic PRRS virus evolving beyond a genome edit call for further genetic barriers preventing or hampering the virus replication.

Genes involved in the interferon response have been identified as potential targets for antiviral targets for a variety of viral infections in humans. Whilst antivirals are not widespread in the livestock industry due to the limited margins, there are opportunities to treat PRRSV with antiviral agents, in particular during infection of pregnant sows, where abortion can lead to the loss of dozens of animals, increasing the cost margin. Therefore, new potential antiviral compounds could be of use to the biotechnology sector.