2012 Annual Report
1a.Objectives (from AD-416):
Objective 1: Identify genetic variations associated with disease resistance to PRRS virus and identify biological determinants associated with anti-PRRS virus host responses to discover genetic and biological determinants associated with disease resistance to infectious diseases of swine.
Objective 2: Discover effective immune intervention strategies to prevent and control infectious diseases of swine by developing immunologic tools to enhance our understanding of swine immune system development and novel assays to evaluate pig responses to infectious diseases; and by assessing differential responses to infection versus vaccination to determine what pathways are associated with protective responses of pigs to respiratory pathogens.
1b.Approach (from AD-416):
Assess whether genomic variation in pigs is associated with differential PRRS resistance/susceptibility and biologic responses to PRRSV infection.
The PRRS Host Genetics Consortium (PHGC) was developed to assess the genomic basis of PRRS disease resistance. The PHGC is a national effort to assess pig resistance/susceptibility to primary PRRSV infection and associated growth effects. Our research will focus on the PHGC objectives:.
1)use genotyping and phenotyping tools to determine if there are host genes that control resistance/susceptibility to PRRSV infection;.
2)verify genetic variation in response to PRRSV, via improved health, survivability and growth; and.
3)identify relative importance of different phenotypes, and their heritability, that predict response to PRRSV infection.
Identify biological determinants associated with anti-PRRS virus host responses.
We hypothesize that there will be functional genomic and proteomic differences that correlate with resistance to PRRSV infections. Using PHGC samples, functional analyses will be performed to identify gene networks and resistance associated biomarkers that differ in high versus low viral load pigs. Our goal would be to identify markers expressed pre-infection, e.g., serum biomarkers in uninfected pigs.
Develop immunologic tools to enhance our understanding of swine immune system development and novel assays to evaluate pig responses to infectious diseases.
A portion of the time devoted to this project will involve research to develop and improve immune reagents available to characterize swine immune responses and regulation. Numerous commercial sources provide reagents for swine immunity research; yet, compared to human and mouse, these resources are limited. Working with the U.S. Veterinary Immune Reagent Network (VIRN) we will develop new immunologic reagents and assays to measure swine cytokines, chemokines, and immune cell surface molecules. These will help identify key features of an effective swine immune response to infectious pathogens
Assess differential responses to infection versus vaccination to determine what pathways are associated with protective responses of pigs to respiratory pathogens. Biomarkers are present in pigs that correlate with protective immunity to respiratory pathogens. Determining whether a response to infection or vaccination is protective or pathologic is essential for the design of new vaccines, diagnostics and therapeutics. For pigs this means understanding the genes and proteins, networks and pathways involved in these responses. Based on the newly finished swine genome sequence, we will identify and annotate some of the numerous genes and regulatory sequences encoded in the swine genome. With that information, functional genomic tools will be applied to assess infection or vaccination responses. Research targets will be infection and vaccination of pigs with respiratory pathogens, particularly PCV2b and PRRSV.
This project investigated swine immunity to viral infectious diseases, targeting the most economically important swine pathogen, Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). With National Pork Board (NPB) funding, ARS scientists at Beltsville, MD, working with scientists at Kansas State University (KSU), Iowa State University (ISU), and commercial pig breeding and animal health companies, established the PRRS Host Genetics Consortium (PHGC). This Consortium has now completed 12 infection trials of 200 commercial pigs each with a goal of determining the role of host genetics in resistance/susceptibility to PRRSV infection and associated growth effects. All PHGC data is stored in the secure, password protected PHGC database at ISU: http://www.animalgenome.org/lunney/index.php Access to the PHGC database is open to Consortium members who have signed a Cooperative Research and Development Agreement (CRADA) Material Transfer Agreement (MTA) #58-3K95-9-1319 organized by ARS. This CRADA MTA was updated in FY12 (#58-3K95-1-1518-MTA). Other grants associated with this effort and with this ARS project have completed single nucleotide polymorphism (SNP) chip typing of all PHGC pigs and the first genome wide association studies (GWAS) that have already mapped several PRRS resistance genes. In fact a major gene on swine chromosome 4 has been identified as accounting for 15% of viral load variation and 11% of weight gain after PRRSV infection. Separate analyses of whole blood RNA and serum are probing for differences in gene and protein expression following PRRSV infection. The PHGC has identified host genes involved in PRRSV resistance or susceptibility, as well as in the ability of some pigs to grow normally despite being PRRSV infected. Results should help pig breeding companies to identify and provide more disease resistant pigs to producers. In addition these studies should provide new alternatives to address immunity to PRRSV infection resulting in improved biotherapeutics and vaccines.
This project has focused on immune studies, targeting the role of cytokines and chemokines in stimulating immune, disease and vaccine responses, assessing gene and protein responses. Major efforts include leading the swine team of the U.S. Veterinary Immunological Reagents Network (VIRN) and using recombinant protein and hybridoma technologies to address the dearth of immune reagents for the pig, as noted at www.vetimm.org. Genomic and protein tools, as well as microarrays and proteomics, initiated through this project are now actively used by researchers worldwide. Validation of the our Fluorescent Microsphere Immune Assay (FMIA), to measure 8 swine cytokines and chemokines simultaneously in pig sera, oral fluids, and cell supernatants, has opened up major routes for examining infection and vaccine responses and predicting protective versus pathologic immune responses.
As part of the PRRS Host Genetics Consortium (PHGC), genome wide association studies (GWAS) were performed to identify genetic determinants of pig resistance/susceptibility to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection by a team led by ARS researchers at Beltsville, MD, (BARC). The primary samples tested are those collected through the PHGC, a national effort to collect phenotypic data to assess the role of genetics in determining pig resistance to PRRSV infection and related pathology and growth effects. DNA samples from pigs from eight PHGC trials and their parents (~2,150 DNA samples total) were prepared at BARC and were genotyped with the 60K single nucleotide polymorphism (SNP) chip, the Porcine SNP60 BeadChip. Heritabilities that influence serum PRRS viremia and weight gain after infection were moderate at 0.30. At Iowa State, extensive GWAS were performed on data from PHGC1-3 data and regions on swine chromosome 4 (SSC4) and SSCX were identified that are involved with viral load, and on SSC1, 4, 7, 17 for weight gain. Genomic estimated breeding values (GEBV) for the SSC4 region were perfectly and favorably correlated at -1, i.e., for the desired effects of decreased virus and increased weight. Thus, pig response to experimental PRRSV challenge has a strong genetic component with a major locus on SSC4 explaining a substantial proportion of the genetic variance. These results could have a major impact in the swine industry by enabling geneticists to develop plans for marker-assisted selection of pigs with improved response to PRRS. This information has been disseminated to swine breeders, genetics companies, and genotyping services so that sets of these recommended genetic markers can be employed in future breeding programs to increase disease resistance.
Swine immune reagents were developed to assist researchers to probe immune responses to infectious diseases and to improve vaccine design. The U. S. Veterinary Immunological Reagents Network (VIRN) (www.vetimm.org) was established to address the dearth of immunological reagents for food animal species. ARS researchers at Beltsville, MD, (BARC), working with Kingfisher Biotech Inc., www.kingfisherbiotech.com/ have expressed several new recombinant swine immune proteins in yeast, including recombinant chemokines (e.g. interleukin-17A (IL-17A) and IL-17F). Bioassays performed at BARC affirmed that the expressed proteins were bioactive. Efforts are underway with our VIRN collaborators to use these proteins for immunizations and monoclonal antibody development. Products generated by the VIRN will be used by animal health researchers, veterinarians, vaccine manufacturers, and other commercial sources.
The U.S. Veterinary Immunological Reagents Network (VIRN) website for swine immunological reagents was expanded and updated. ARS researchers at Beltsville, MD, have provided all documentation and knowhow for the USDA/NIFA/AFRI grant supported immune toolkit or VIRN website (http://www.umass.edu/vetimm/swine/index.html) swine pages. They scanned commercial websites and publications for swine immune reagents and organized the data into easily accessible tables of currently available reagents for swine immunology. This identified some problems with previously available reagents being pulled from the market. This list served as the basis for updating the swine priorities for developing new VIRN immune reagents and for VIRN colleagues and swine collaborators to develop plans for their production. The ARS researchers also posted all current results and updated protocols on the VIRN website. As a result, this website enables researchers worldwide to quickly identify available immune tools and protocols for their experiments. Overall swine disease and vaccine researchers now have improved tools to address pig health and vaccine design.
Swine innate immune reagents are desperately needed to help scientists probe immune responses to infectious disease threats and to improve vaccine design. The early innate period of immunity is crucial for stimulating protective and preventing pathologic immune responses. ARS researchers at Beltsville, MD, (BARC), working with Kansas State University scientists have expressed swine type 1 interferons (IFNs), specifically, several IFN-alphas (IFNa1, IFNa6, IFNa9) and IFN-beta, using mammalian cells. These products were transferred to Cornell University to prepare monoclonal antibodies (mAbs) to these unique IFNs. To date several panels of mAbs have been screened at BARC and found to be reactive with all IFNas. Alternate immunization protocols are now being pursued to target mAb reactive with only one IFNa gene product so their effects can be discriminated. Progress for these efforts has been documented on the Veterinary Immunological Reagents Network (VIRN) swine website. Products generated by this grant and by VIRN will be used by animal health researchers, veterinarians, vaccine manufacturers, and other commercial sources to improve research on responses to infectious disease outbreaks and to target new pathways active in protective immune responses.
New multiplex methods have been developed to measure important immune proteins, termed cytokines and chemokines, in pig blood, serum, and oral fluids. ARS researchers at Beltsville, MD, (BARC), worked with university partners to develop a multiplex bead based swine cytokine assay, termed a Fluorescent Microsphere Immune Assay (FMIA), to measure eight cytokines simultaneously in pig serum. A 'multiplexed' assay is advantageous because it measures multiple cytokines simultaneously and requires only a small volume of each sample. The assay detects cytokines that regulate several aspects of the swine immune system: the early innate response; the regulatory, the T helper 1 and T helper 2 cytokines. The assay was tested on sera collected from porcine reproductive and respiratory syndrome virus (PRRSV) challenge studies as part of the PRRS Host Genetics Consortium effort. Initial observations indicate differences in serum immune protein responses early after PRRSV infection. Interferon-alpha levels remained statistically higher in high versus low PRRSV burden pigs; this correlated with the poorer control of immunity during PRRSV infection. This assay provides a major new tool to define protective immune responses in PRRS and other respiratory diseases in pigs and importantly for designing more efficacious vaccines for swine pathogens.
A high quality draft reference pig genome sequence has been generated. In preparation for this a group of scientists interested in swine immunology formed the Immune Response Annotation Group (IRAG). More than 30 scientists internationally, including ARS researchers at Beltsville, MD, (BARC), worked closely with the Havana group at the Wellcome Trust Sanger Institute on the community annotation of immunity associated genes in the pig genome. This resulted in the detailed manual annotation of those genes which constitute 5% of the pig genome. The information developed by IRAG was summarized and included as part of the final sequence. This annotation work expanded swine bioinformatic capabilities for immune response associated genes, including identifying rare splice variants found during gene expression studies. Results from gene expression studies should help animal health researchers identify new pathways involved in disease prevention, control and pathology. Probing these pathways will lead to alternate vaccine targets and disease therapeutic approaches.
The PRRS Host Genetics Consortium (PHGC) has now completed 12 infection trials to assess the role of host genetics in PRRSV resistance. With ARS, National Pork Board (NPB), and Genome Canada funding, ARS scientists at Beltsville, MD, (BARC) are working with PHGC collaborators to collect samples for a detailed phenotype of the role of pig genetics in resistance to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection. Using a nursery pig PRRSV infection model, 12 infection trials of 200 pigs each have been completed, thus well surpassing the original goal of testing 1,500 pigs. High health pigs donated by commercial breeding companies were infected with a 1998 PRRSV isolate for the first nine trials; whereas a more recent, 2006 PRRSV isolate was used for PHGC trials 10-12. For every pig, blood was collected at 10 timepoints. This included 20,000 samples each of sera (for virus and immune proteins), and whole blood Tempus tubes (for RNA), as well as tonsils (for virus persistence tests), and ears (for DNA). At BARC, genomic DNA samples were prepared and the single nucleotide polymorphism (SNP) genotyping completed for all PHGC 1-8 pigs. All PHGC data is being stored in the secure, password protected PHGC database at ISU: http://www.animalgenome.org/lunney/index.php. Other grants associated with this ARS project have funded analyses of whole blood RNA and serum for differences in gene and protein expression after infection. The PHGC and related projects will help identify the role of host genes in PRRSV resistance or susceptibility as well as in the ability of some pigs to grow normally despite being PRRSV infected. This project has already revealed new genetic regions that are associated with PRRS resistance or alternately with increased PRRS susceptibility. Results should help pig breeding companies to identify and provide more disease resistant pigs to producers.
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