Engineering resistance to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

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Porcine Reproductive and Respiratory Syndrome (PRRS) is a viral disease of pigs that causes major economic losses. 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 ca.1/3 of the cost of infectious disease to the US pig industry, ~$600M p.a.. PRRS is the most costly disease to pig industries of Europe and North America and new PRRSV variants have the potential to be even more devastating. PRRSV infects subpopulations of differentiated macrophages, with alveolar macrophages being the major target cells.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. Thus, there is scope for genetically improving traits related to the capacity of pigs to cope with PRRS infection and disease at the innate immune level. However, breeding for disease resistance is constrained by the nature of the available 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 increased efficiency and sophistication of methods to genetically modify farmed animals offers alternative approaches to generate animals which are genetically resistant to specific pathogens. The approaches to engineering resistance to a viral pathogen, such as PRRSV, include interfering with the receptor(s) through which the virus gains entry, for example by ablating the receptor or over-expressing a soluble form of the receptor which could bind the virus and blocks its entry.Recent studies have revealed the molecular mechanisms through which PRRSV enters macrophages during infection. The macrophage specific scavenger receptor cysteine-rich (SRCR) CD163 has been shown to be a key role in these processes. Cells which are refractory to PRRSV infection can be converted to a PRRSV permissive state by the addition of transgenes expressing CD163. Treating susceptible porcine alveolar macrophages with anti-CD163 antibodies reduces PRRSV infection in a dose-dependent manner. There is evidence to indicate that the SRCR domain 5 of the CD163 protein is the key component involved in PRRSV entry and release.The aim of this project is to test the hypothesis that cells and pigs can be engineered to be resistant to infection with PRRSV by genetic modification of the CD163 gene. We propose three strategies to engineer resistance to PRRSV: knocking out the CD163 gene; knocking out the SRCR domain 5 of the CD163 gene and over-expressing a soluble form of the extracellular domains of CD163.