Location: Animal Parasitic Diseases Laboratory2012 Annual Report
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.
3. Progress Report:
This functional genomics project uses samples collected from pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV), a major swine pathogen causing losses to the U.S. pig industry of $664 million per year. The goal is to determine which anti-viral response pathways differ in PRRS-resistant versus PRRS-susceptible pigs using samples collected as part of the PRRS Host Genetics Consortium (PHGC). ARS Researchers at Beltsville, Maryland (BARC) have partnered with Washington State University (WSU) scientists to use PHGC blood samples to assess gene expression responses. To date, sets of blood RNA samples have been prepared at BARC from 120 pigs from three different PHGC trials. Michigan State University (MSU) scientists have used the Swine Protein-Annotated Oligonucleotide Microarray, or Pigoligoarray (www.pigoligoarray.org), to evaluate changes in gene expression of blood RNA from 12 pigs collected at 0, 4, 7, 11, 14, 28, and 42 days post infection (dpi). Biostatistical and bioinformatic analyses have been completed at MSU to identify genes and pathways that are associated with pigs that clear the PRRSV and that grow well despite PRRSV infection. Analyses of data from this first set of arrays has resulted in the decision to focus on 0, 4, and 7 dpi samples so that gene expression of early anti-PRRSV infection can be evaluated in a more robust statistical manner. Targeted quantitative polymerase chain reaction (PCR) assays will help to affirm which genes are correlated with viral load and/or weight gain by comparing the (1) most desirable, PRRS-resistant, low virus, high weight gain pigs with the (2) worst, PRRS-susceptible, high virus, low weight gain pigs. At WSU, comparative bioinformatic tools are being used to annotate specific porcine genes and their splice variants. Quantitative PCR assays were developed to determine whether splice variants are differentially expressed in samples from PHGC populations with high or low viral load and/or weight gain. If abundantly expressed splice variants can be identified they may serve as key biomarkers of anti-PRRS responses.