Identification of Genetic Variation in Innate Immune Response Genes Associated with Resistance to Chicken Viral Infections

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. <P>
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. <P>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. <P>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.