Award details

Understanding the CD163 - PRRS virus interaction to improve genetic engineering for resistance

Reference	BB/R004463/1
Principal Investigator / Supervisor	Dr Christine Tait-Burkard
Co-Investigators / Co-Supervisors
Institution	University of Edinburgh
Department	The Roslin Institute
Funding type	Research
Value (£)	530,236
Status	Completed
Type	Research Grant
Start date	01/12/2017
End date	31/08/2021
Duration	45 months

Abstract

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. The macrophage cell surface protein involved in PRRSV fusion was found to be CD163. Out of the nine scavenger receptor cysteine-rich (SRCR) domains that form a pearl-on-a-string structure as the extracellular part of CD163, SRCR domain 5 (SRCR5) was found to be essential for PRRSV's ability to infect the host cell. Recent work by me and others demonstrated that genome editing can be a viable tool to combat PRRSV infection and confirmed the importance of CD163 in PRRSV infection. Work by another research group has shown that pigs in which the CD163 gen has been knocked out are resistant to PRRSV infection. However, the expression and function of CD163 in these animals is completely abolished and could have negative effects on the animals. My work removed SRCR5 by deletion of the encoding exon from the pig genome. I found that macrophage cells from these animals were not only resistant to PRRSV infection but retained expression of the truncated deltaSRCR5 CD163 on the macrophage surface, preserving the haemoglobin-haptoglobin scavenger function of the protein. The aim of this project is to further assess the CD163 - PRRSV interaction and the potential of the PRRSV to adapt to a deltaSRCR5 pig as well as to generate even more subtle changes to the pig genome than exist in the current deltaSRCR5 pigs. I propose the following strategies to address these questions: to identify domains and amino acids of CD163 interacting with PRRSV; to assess the PRRSV-CD163 signalling and interactome; to serially passage PRRSV on deltaSRCR5 to mimic virus evolution; to produce deltaSRCR5 pigs by exon skipping using CRISPR/Cas9 homology directed repair.

Summary

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. 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. One of these host proteins, CD163, has been found to be essential for the virus' escape from the uptake vesicle during the entry process by membrane fusion. CD163 is expressed on the surface of macrophages and the extracellular portion of CD163 forms a pearl-on-a-string structure of nine scavenger receptor cysteine-rich (SRCR) domains. SRCR domain 5 (SRCR5) was found to be essential for PRRSV's ability to infect a host cell. In addition to mediating PRRSV infection, CD163 has a variety of important functions within the host cell, such as removing haemoglobin-haptoglobin complexes from the blood, thereby preventing the formation of reactive oxygen species that could damage other cells. My recent research together with others demonstrated that genome editing is a viable tool to combat PRRSV infection and confirmed the importance of CD163 in in this process. Work by another research group has shown that pigs in which the CD163 gene was knocked-out are resistant to PRRSV infection. However, the expression and function of CD163 in these animals is completely abolished and thus their ability to remove haemoglobin/haptoglobin complexes from blood, ischaemic repair and inflammation response may be impaired. My work removed SRCR5 by deletion (deltaSRCR5) of the encoding exon from the pig genome. I found that macrophage cells from these animals were not only resistant to PRRSV infection but also expressed the truncated deltaSRCR5 CD163 on the macrophage surface whilst maintaining haemoglobin-haptoglobin scavenger function. It is expected that removal of SRCR5 and thereby destruction of the "lock" abrogates the key-lock interaction of PRRSV with its receptor. Thereby, it prevents fusion of PRRSV with the host cell membrane. However, we do not understand enough of the CD163-PRRSV interaction to exclude the potential for this highly mutagenic RNA virus to adapt to a deltaSRCR5 cell or pig. Therefore, we need further understanding of the CD163 - PRRSV interaction and insight into PRRSV's ability to adapt to the deltaSRCR5 modification. My research on deltaSRCR5 shows that removal of a single exon from CD163 can abolish PRRSV infection, emphasising the great power of genome editing technology to induce subtle genetic changes. The aim of this project is to further assess the CD163 - PRRSV interaction and the potential of the PRRSV to adapt to a deltaSRCR5 pig as well as to generate even more subtle changes to the pig genome than exist in the current deltaSRCR5 pigs. I propose the following strategies to address these questions: to identify domains and amino acids of CD163 interacting with PRRSV; to assess the PRRSV-CD163 signalling and interactome; to serially passage PRRSV on cells from deltaSRCR5 pigs to mimic virus evolution; to produce functional deltaSRCR5 pigs by exon skipping rather than exon deletion.

Impact Summary

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. 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. The recent demonstration that such genetically-engineered, PRRSV-resistant pigs can be generated shows the potential impact of such pigs for the pig industry. Further research into these animals is thus not only timely but also necessary to assure breeders and consumers alike that genetically-engineered pigs do not present a long-term risk for virus evolution. 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.

Committee	Research Committee A (Animal disease, health and welfare)
Research Topics	Animal Health, Immunology, Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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