1a.Objectives (from AD-416)
The specific objectives of this agreement include Identify differentially expressed (DE) genes in blood in response to PRRSV infection; Determine putative gene sets and pathways that predict a pig's ability to clear PRRSV infection and maintain weight gain; and Validate utility of gene sets and pathways for prediction of responsiveness to PRRSV infections in multiple populations. Predictive blood tests of pigs with improved PRRS disease resistance and growth maintenance; increased understanding of mechanisms involved in pig responses to PRRSV infection; scientific publications.
1b.Approach (from AD-416)
Washington State University (WSU) will contribute to the first and third objectives. The first objective WSU will search public databases and use bioinformatic tools to examine the transcriptome of alveolar macrophages in response to PRRSV infection. This data will serve as a reference to determine what genes are expressed exclusively or predominantly in response to PRRS infection and compared to Michigan State University full transcriptome data. For the third objective, WSU will annotate specific porcine genes manually using comparative annotation procedures for retrieval of both cDNA and genomic DNA sequences of each gene identified in the second objective. WSU will then detect putative splicing forms in each of selected DE pig genes using three approaches – comparative analysis in mammals, EST evidence and PCR screening. The different sizes of products will be sequenced and the tentative splicing forms examined. Primers will be designed to target different splicing form and RT-PCR carried out to examine which form is most abundantly expressed in high or low responders to PRRSV infection.
A new project was established to perform functional genomic analyses to determine response pathways that differ in porcine respiratory and the reproductive syndrome (PRRS) resistant versus susceptible PRRS Host Genetics Consortium (PHGC) pigs. ARS Researchers at Beltsville, MD have partnered with Washington State University scientists to use PHGC samples to assess whole blood gene expression responses to identify genes and pathways that are associated with pigs that clear PRRS virus (PRRSV) and that grow well despite PRRSV infection. With Michigan State University scientists multivariate statistical analyses of viral load and weight data have categorized PHGC pigs into 4 extreme categories including the most desirable, PRRS resistant low virus/high weight gain pigs, and the worst, PRRS susceptible high virus/ low weight gain pigs. This statistical categorization of pigs from each PHGC trial provides a critical basis for selecting pigs and samples for detailed analyses of processes that control transcriptional responses to PRRSV infection. At WSU comparative bioinformatic tools will be used to annotate specific porcine genes and their splice variants and determine whether they are differentially expressed in samples from specific PHGC populations. If abundantly expressed splice variants can be identified they may serve as key biomarkers of anti-PRRS responses. As this work progresses we expect to develop predictive gene expression pathways and classifier genes that identify pigs which resist PRRSV infection and grow normally.
Disseminating progress on this grant was through regular email and phone contact, scheduled conference calls and yearly Consortium meeting with the participating labs discussing project plans, experimental design and reviewing data and presentation options.