Genome-wide mapping and identification of genes controlling resistance to Marek's Disease virus infection in commercial layer chickens

Marek's Disease (MD) is caused by a highly contagious virus (MDV), which is a continuing threat to our global poultry industry and food security with estimated losses of £1-2 billion per year. These losses are associated with the cost of the disease, due to increased mortality, escalating vaccination costs and reduced egg production. It is therefore a disease that has an impact on our economy and ability to compete internationally, with additional concerns for animal health and welfare, and our global food security. Although it has been controlled by vaccination for over 40 years, there is growing evidence from researchers that intensive use of vaccines is driving the virus to have an increasing ability to kill poultry, with more aggressive strains of the disease continuing to emerge each year.

An alternative means to control this disease is being sought through breeding birds with enhanced resistance to MDV. Such differences were first reported nearly 80 years ago. Subsequently, genes associated with the major histocompatibility complex (MHC), which plays an important role in the immune system, were shown to contribute towards resistance to MDV. Despite this, other genes also have a strong influence on MDV resistance. This has been determined most effectively from studies using inbred strains of chickens that have identical MHC but still show differences in their susceptibility towards MDV.
Many chromosomal regions that control MDV resistance have been defined (quantitative trait loci, or QTL) but very few genes have been verified as controlling the host response towards this disease, which is typical for most complex traits. Those which have been identified include growth hormone (GH1), the cytokine SCYC1 and the cell surface marker on lymphocytes LY6E. Identifying genes involved in resistance (or susceptibility) to MDV and resolving their role is crucial to understanding this disease and why it appears to becoming more fatal. This knowledge is important; providing the means to control and reduce this threat to the poultry industry.

This proposal is extremely timely in that we now have at our disposal many new research tools which may enable us to identify genes involved in resistance to MD infection. These include QTL data, access to MDV challenge facilities, genomic sequences of chicken lines, a large QTL high resolution mapping population (now in its 6th generation) based on parents differing in their resistance to MDV and a panel of 600K SNPs (single nucleotide polymorphisms - genetic markers) which can be used together to fine map Marek's Disease QTL to high resolution to genomic regions containing around 25 genes. This, in conjunction with high throughput sequencing and bioinformatics approaches, will allow us to not only predict candidate genes for MD resistance but also possible causative mutations within these genes.

We also have available to us all the MDV proteins cloned into suitable vectors for testing in 'yeast-2-hybrid' experiments for direct protein/protein binding between viral and host proteins. In this way we will be able to identify genes which bind directly with viral proteins and then go on to test whether mutations within these genes alter any binding interactions.
In this proposal we aim to determine the chromosomal regions that play a role in MD resistance in commercial egg-layers, identify genetic variation within candidate genes, explore direct protein binding interactions between host and viral proteins, and examine the functional consequences of genetic variation in these genes.

The outcome of this project will be the identification and characterization of valuable genetic markers in commercial egg-layer lines for use in selective breeding programs within the poultry industry, which would result in large economic and health benefits. This comes at a time when there are renewed pressures on the poultry industry as the world population expands and concerns over food security increase.

We will use a full-sib intercross line created for fine mapping QTL for MD. 1200 F6 animals, selected for high or low resistance from 2400 tested will be genotyped with 600K SNPs, expected to map QTL to regions of about 5cM. Genome sequences of 10 founder parents will be determined and used to impute the sequence of QTL containing regions (QTLR) in F6 animals.

We will annotate QTLR for genes based on Ensembl and RNAseq resources. Sequences will be examined for genetic variants, such as SNPs, indels, simple sequence repeats and CNV. Functional effects of variants on genes and other constrained sequences will be predicted using bioinformatics tools.

We will annotate variants that affect regulation of genes as shown by allele-specific-expression, based on unequal number of RNAseq reads mapped to alternate alleles. Gene regulation will be based on three comparisons: (i) macrophage LPS/control, (ii) macrophage MDV/control or (iii) spleen MDV/control. SNPs with a significant effect for any of these responses will be selected for further investigation.

Candidate host proteins will be tested for binding to MDV proteins via Y2H screens against a library of 100 MDV genes sub-cloned into pGADT7, and verified by co-immunoprecipitation in chicken splenocytes. Alternate alleles of host proteins may show altered binding to MDV proteins these will be tested by semi-quantitative co-immunoprecipitation.

Candidate functional SNPs for MD resistance will be tested for their utility in 8 pure-bred lines that serve as parental lines for Hy-Line layer crosses. This will be based on large numbers of males progeny tested for MD resistance within each line for the past 15 years. Tests for SNP-trait association will be in two stages. Stage-1 based on pooling resistant and susceptible individuals within each line and Stage-2 based on selective individual genotyping of SNPs that achieve suggestive statistical significance in Stage-1.

The UK poultry industry faces numerous challenges in order to remain sustainable. These include the imminent move to more extensive rearing systems; the withdrawal of prophylactic and many therapeutic antibiotics, and other drugs such as anti-coccidials; resistance and residue problems with anti-helminthics. These challenges will all have an impact on poultry health and the potential to impact on human health. For example, with an increased incidence of zoonotic pathogens in chickens, there is the potential to lead to an increase in these diseases in human. It is important that poultry breeders are able to select for genetic improvement in performance when birds are reared in such environments. New and effective disease control requires detailed understanding of host-pathogen interactions.

Marek's Disease (MD) is caused by a highly contagious oncogenic alpha-herpesvirus which is a continuing threat to the world's poultry industry, with estimated losses of £1-2 billion per annum. Although it has been controlled by vaccination for over 40 years, there is growing evidence that intensive use of vaccines is driving the virus to increasing virulence and new, more virulent strains continue to emerge. An alternative means to control MD would be through the selection of birds with enhanced genetic resistance to the virus (MDV). Several potential MD resistance QTLs have been identified, but relatively few genes have been associated with the disease.

This project directly addresses this problem, and should result in a wealth of knowledge regarding pathways, genes and genetic mutations involved in MD disease resistance. It should also be possible to patent these candidates for use as selective markers in breeding programmes. Beneficiaries of this research include academics interested in the genetics of disease resistance, host-pathogen interactions and the innate immune system. Results will be published, when appropriate, in peer-reviewed scientific journals and presented at international conferences.

Results of this research will have potentially far-reaching social and economic consequences. A reduction in the incidence and losses caused by MD through selective breeding would have an important economic impact on the poultry industry in the UK and around the world. Selection could be done directly on genes, thus improving selection accuracy and decreasing the need for costly challenge studies and unnecessary loss of birds. Improvement in disease resistance at the primary breeder level will have a positive impact on the well-being of hundreds of millions of birds at the commercial production level. The health and well-being of farmed chickens would be greatly improved and a major source of human food production would be further secured.

Established contacts with companies such as Pfizer Animal Health, Aviagen and Hy-Line will ease this transfer of knowledge from the academic environment to industry. Markers will have been detected and tested in existing commercial lines and would therefore be ready for utility by poultry breeders. Effective uptake of the outcomes of our research is facilitated by close links with Biosciences KTN, who identified disease resistance and animal health as the top priorities for UK breeding industries. This work has the potential to impact on the sustainability of the poultry industry in the UK, and thus to inform DEFRA policy.

In scientific terms this project falls within the remit of the BBSRC 'Animal systems, health and wellbeing' Research Committee and matches the BBSRC scientific priority of 'Food Security' in terms of 'Animal Health' and 'Livestock Production'.