Identification of genetic variation in innate immune response genes associated with resistance to chicken viral infections

The UK poultry industry faces increasing challenges in the 21st century. Many of the drugs used to control diseases in poultry are being withdrawn from use. At the same time, improving welfare standards mean that the industry is becoming more free-range. But the downside is that free-range chickens face higher rates of disease challenge, for example through contact with wild birds. One answer to this problem is to breed birds, which have a higher natural resistance to disease. To do this, we need to understand the bird's immunity to disease, so that we can identify ways to control that response. We already know that in birds and mammals the immune response can be divided into two arms - the innate response and the adaptive response. The latter is a very specific response, which hopefully leads to immune 'memory', in other words the ability to respond rapidly to subsequent infections with the same pathogen. This is how vaccination works, however, it is specific to each pathogen the bird encounters, and thus would not give general resistance to disease. Our target is the innate immune response. This is a generalised response that recognises components of a wide range of pathogens, for example, specific types of molecules in a bacterial coat. It serves two roles - it limits infection until the adaptive immune response can kick in, and also provides signals that control the adaptive immune response. For many infections, a strong innate immune response is all that is needed to control the pathogen and prevent the onset of disease. Our initial aim in this project is to identify as many of the genes in the chicken as possible that are involved in the innate immune response. This is now possible since the chicken genome was sequenced last year and already almost 18,000 genes have been predicted. Our initial gene set will be determined by comparisons with similar gene sets in mammals, and what we can identify from the chicken genome sequence. The next step will be to identify which members of this predicted set of genes are actually involved in the innate immune response to infection with viruses. We will do this by infecting birds with different viruses and looking at the expression of genes following infection, on a global level using whole genome microarrays. These are glass slides containing short unique sequences corresponding to each known gene in the chicken genome that can be used to detect whether a gene is turned on or off. After complex mathematical analysis, one can examine global gene expression using these microarrays and identify which chicken genes respond to a particular viral infection. We have chicken lines, which we know differ in their resistance to viral infections. We suspect that at least some of that difference will be at the level of the innate immune response. Once we know which genes are involved in the innate response to viral infection, we will then look for sequence differences or polymorphisms between these chicken lines. We will be looking for changes at single nucleotides within the DNA sequence of these genes, known as SNPs (single nucleotide polymorphisms). We will then predict, and later test, if these SNPs are likely to make a functional difference to the protein encoded by the gene, either in its structure (for example by changing the amino acid sequence) or the level of its expression. Finally, we will then assess if individual SNPs track with resistance to viral infection using crosses between resistant and susceptible lines, in simplest terms by seeing if the SNP is always found in a resistant bird. These SNPs can then be used as selectable markers in normal breeding programmes to select birds that are naturally more resistant to viral infection.

The UK poultry industry faces many challenges to remain sustainable, including moves to more extensive rearing systems; withdrawal of antibiotics and other drugs such as anti-coccidials; resistance and residue problems with anti-helminthics. These will all impact on poultry health, but also the potential to impact on human health. Increased incidence of zoonotic pathogens in chickens, such as avian influenza and SARS, could lead to an increase in these diseases in man. It is vital that poultry breeders can select for genetic improvement in resistance when birds are reared in such environments. This proposal seeks to identify genes controlling variation in innate immune responses; define the extent of genetic variability in these loci and relate this to resistance/susceptibility to viral diseases; provide new markers for selective breeding for improved innate resistance to viral disease and hence improved food safety. Inbred lines of chickens maintained at IAH differ greatly in their susceptibility to a wide spectrum of pathogens, including viruses. Differences in innate immune responses determine resistance to infection to Salmonella in different broiler lines. It is thus a reasonable hypothesis that a similar mechanism will influence resistance to viral infections. The availability of genome sequences, for both the pathogens under study (Marek's disease virus (MDV), Infectious Bursal Disease Virus (IBDV) and Infectious Bronchitis Virus (IBV)), and also now for the chicken, represents a major shift in our ability to understand host-pathogen interactions. From the chicken genome sequence and existing information on relevant mammalian and chicken genes, we will define a comprehensive set of chicken innate immune response genes (preliminary analysis has identified almost 400). Challenge experiments with the three viruses using inbred lines with varying degrees of resistance to them will be carried out. RNA samples will be used in high-throughput gene expression experiments using Affymetrix chips (containing genes from the chicken and the three pathogens) to identify the subset of innate immune response genes that are involved in the subsequent response. Most amino acids within proteins are under selective constraints with estimates of selection (dN/dS) values around 0.05-0.10. However genes involved in host-defense evolve more rapidly than other genes and have larger dN/dS ratios, probably due to positive selection on specific amino acids as a consequence of the host-pathogen 'arms-race'. Orthologous sequences will be isolated from turkey, duck and Zebrafinch cDNA libraries and compared with chicken genes to estimate dN/dS ratios. Single nucleotide polymorphisms (SNP) within these genes between the inbred lines will be determined by direct sequencing. For coding sequences we will sequence cDNAs from tissues isolated from inbred lines. Sequence alignment between the avian species listed above will be used in combination with SNP and other structural data to assess if a SNP is likely to be functional (i.e. likely to affect the function of the protein). For promoter polymorphisms we will sequence 500-bp of 5'-flanking region. SNP locations will be mapped onto conserved regions and likely transcription factor binding sites to predict possible functional significance. SNP assays will be established to determine segregation of these SNPs with resistance in relevant backcross populations. For MDV and IBDV the relevant DNA panels are already archived from previous experiments with associated phenotypic information. For IBV the relevant backcross population and challenge study will be carried out as part of this proposal. The impact of any segregating SNPs will then be determined with reporter gene and real-time quantitative RT-PCR assays. As a direct output, we expect to identify SNPs correlating with resistance to viral infections that can be used as selectable markers in conventional breeding programs.