Host factors controlling foot-and-mouth disease virus (FMDV) replication: towards genetic control of FMD in pigs

One of the world's biggest challenges is to meet a growing demand for food. Pork is the most widely consumed meat and the world's largest source of animal protein. Foot-and-mouth disease virus (FMDV) causes an economically devastating disease of pigs and other livestock and affects both the developed and developing world. Therefore improved control of FMD in pigs could sustainably improve the performance of global livestock industries and have enormous economic and social value worldwide especially in developing regions such as parts of Asia with rapidly growing demand for pork. FMD vaccines are available and often used to help control the disease in cattle, however such vaccines have limited effectiveness in pigs.

Genetic engineering of animals for disease resistance is a potential alternative strategy for preventing FMD in livestock species. Such engineering is now possible in the domestic pig, as demonstrated by the recent production of gene-edited pigs which are resistant to another unrelated viral pathogen, porcine reproductive and respiratory syndrome virus (PRRSV). Unlike transgenic approaches, gene editing does not add foreign genetic material and this distinction has already allowed gene-edited plants to be used for food production in some regions of the world.

For viruses to replicate inside host cells, they must interact with various host proteins which are in turn encoded by specific genes in the host genome. Genes identified as essential for virus replication but non-essential for normal host phenotype, can be modified or made non-functional so that the virus can no longer replicate. We have preliminary data showing that such genes can be identified in pig cells and that reducing expression of these genes does indeed prevent virus replication. We now wish to i) expand the search for these genes using techniques tailored to the porcine genome, ii) obtain proof-of-principle that disrupting gene expression will prevent FMD in animals using mouse models and iii) generate engineered cell lines to understand how best to prevent viral replication by gene editing of the porcine genome.

We believe the outcomes of the proposed study will provide the information required to begin genetic engineering of the pig for resistance to FMD.

This work will short list host genes essential for FMDV replication in porcine cells in vitro and in knock out mice in vivo, in preparation for studies of gene editing in pigs for resistance to FMD.

Validation of existing target genes (identified with human siRNA): will use porcine specific siRNA transfected into porcine SK-RST cell line followed by transfection of a FMDV sub-genomic GFP replicon or infection with virus engineered to express reporter gene and level of reporter in cells used to quantitate viral replication.

Identification of additional target genes using a porcine-specific CRISPR knock out library: The porcine SK-RST cell line will be transduced with lentiviral libraries containing six sgRNAs against every gene in the porcine genome and then infected with FMDV reporter virus and selected by FACS into low, medium or high level replication populations based on fluorophore signal. High throughput sequencing will correlate occurrences of a sgRNA (each of which targets a specific gene) with viral replication.

Validation of target genes in vivo using FMDV infection in knock-out mice: Knock out murine embryonic fibroblasts (MEF) will be tested prior to animal work. Gene knock out and wild type mice will be inoculated with FMDV in groups of 6 and clinical signs monitored twice daily and extent of viraemia measured post inoculation.

Development of gene editing reagents for knockout of target genes in pig cells and testing the effect on FMDV replication in vitro: Guide RNAs for each gene will be transfected into SK-RST cells together with recombinant Cas9 and tracrRNA. Cells will be cloned and analysed by sequencing and western blot and transfected with GFP-replicon or infected with live virus as described above.

Investigating the barrier to viral adaptation will use serial passage and deep sequencing of virus in gene knock out cell lines to look for evidence of viral adaptation to overcome the gene knockout.

Beyond the academic scientific community, the proposed research may also realise tangible benefits of a social and economic nature. This work could lead on to the development of pigs engineered to be resistant to foot-and-mouth disease and this could have enormous global impact in the future.

The work will also help to establish the use of genome wide CRISPR library screening for livestock species generally and therefore contribute to maintaining the UK at the cutting edge of genetic approaches that contribute to future food security.

These outcomes would be of benefit to Pirbright, Roslin and Edinburgh (and various other UK HEI collaborators on this and other projects), the BBSRC and its stakeholders such as Defra, the UK farming industry and international organisations involved in control of FMDV (and other veterinary pathogens) such as The Food and Agriculture Organization of the United Nations, The European Commission for the Control of Foot-and-Mouth Disease and the World Organisation for Animal Health.

The research will have general impact with the wider scientific community, veterinary and medical practitioners, students and the general public. Engagement with these diverse groups will be achieved via meetings, articles in the trade press, tailored web pages, press releases to the media and travelling shows and outreach events in schools. This will be especially important in the context of genetic engineering of livestock, a potentially controversial subject and where this project could provide a positive example of the benefits to be gained.

If the proposed studies are successful, additional funding will be sought from relevant funding agencies and commercial sources for further research to begin gene editing of pigs for disease resistance. There is extensive experience within the host institutions of patent applications and commercialisation; especially at Roslin with regard to commercial involvement in the development of engineered livestock. Such opportunities will feed into an established system for technology development and knowledge transfer by the Pirbright and Roslin Business Development groups.