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

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.

Porcine Reproductive and Respiratory Syndrome (PRRS) which is a viral disease of pigs is the most costly disease to the pig industry. PRRS virus (PRRSV) which is a rapidly evolving small enveloped RNA virus infects subpopulations of differentiated macrophages, with alveolar macrophages being the major target.
The increased efficacy of genetic modification methods offers approaches to generate animals which are genetically resistant to specific pathogens. In particular, genome editing technology using Transcription Activator-Like Effector Nucleases (TALENs) injected directly into zygotes enables efficient and precise modification of animal genomes, including the pig. 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 or mutating 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 have 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.
We aim 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.

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, including our collaborators at Recombinetics Inc.

How will they benefit from this research?
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. Thus, the development of novel and/or more effective strategies to control PRRS will improve the sustainability of the pig industry and potentially reduce 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. With increasing capabilities to genetically modify farmed animals there are opportunities to engineer resistance. It is now timely to explore the opportunities for engineering pigs for enhanced resistance or tolerance to PRRSV infection. The interaction between host (pig) and pathogen (PRRSV) are better understood and new genome editing technologies facilitate the necessary engineering.

Public acceptance of genetically modified animals remains uncertain, especially in Europe. However, the development of non-transgenic pigs engineered for enhanced disease resistance using genome editing technology, which introduces no exogenous DNA, has the potential to re-shape the debate. Moreover, given the impact of PRRS in key markets such as North America and China where genetically edited animals are potentially acceptable, beneficial impacts could be delivered to the pig industry within 3-5 years of project completion.

The participation of Genus plc, the leading global supplier of genetically improved germplasm to the pig industry, will greatly facilitate the realisation of the project impact in the pig breeding sector. Through the activities of the COST Action FA902 on "Understanding and combating porcine reproductive and respiratory syndrome in Europe" [EuroPRRS] chaired by the coI (TAA) we have excellent links to the animal health sector with interests in PRRS.

For Recombinetics Inc. the benefits would include exemplification of their licensed genome editing technology.