Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 6/24/2008
Publication Date: N/A
Technical Abstract: Our goal is to uncover genetic components involved in early immune responses during porcine reproductive and respiratory syndrome virus (PRRSV) infection. PRRS costs U.S. swine producers >$700 million annually. We want to determine what are the most significant pathways and genes involved in early immune responses following PRRSV infection as compared to those after protective vaccination. Since innate immune responses are weak after PRRSV infection we hope to understand which genes are differentially stimulated in the vaccinated versus the PRRSV challenged pig as a guide to determining effective immunity against this infection. We used the Swine Protein-Annotated Oligonucleotide Microarray (www.pigoligoarray.org), a second generation porcine 70-mer oligonucleotide-microarray comprised of 20,400 oligos. Data on validation of the pigoligoarrays was developed as a collaboration between several institutions and supported by the US Pig Genome Coordination Program. Transcriptional profiles were analyzed for TBLN, lung and longissimus dorsi muscle from normal pigs and confirmed using quantitative real-time PCR (Q-PCR) for 12 selected candidate genes. Our diagnostic analyses, using pigoligoarray designed control features including negatives and perfect match/mismatch oligonucleotide sets, revealed no evidence of non-specific hybridization. Comparative hybridization of these control tissues affirmed the tissue specific gene expression patterns which was confirmed by Q-PCR. Statistical analyzes affirmed highly significant differences in gene expression between genes and tissues. Our goal was to use this pigoligoarray to identify immune regulatory and protective pathways. In our initial PRRS experiment animals were divided into three groups: (1) pigs infected with a virulent PRRSV isolate MNW2B; (2) pigs vaccinated using a contemporary PRRS ATP vaccine (Ingelvac®); and (3) control pigs. Tissues [cranial lung, distal lung, tracheobronchial lymph nodes (TBLN) and tonsils] were collected between days 3-6 post infection/vaccination. Total RNA was isolated and labeled using Alexa Fluor® 555 and Alexa Fluor® 647 dyes (Invitrogen). A microarray loop design was applied to compare gene expression between individuals from all three groups and additionally to investigate differences due to day post infection within the PRRSV infected group. Analyzes were carried out using R and SAS software. Results included three data sets comparing the different groups of pigs: (1) Vaccinated verses Control; (2) Vaccinated versus Infected; (3) Control versus Infected. Data from all animals representing the different groups were included in the statistical analyses.Network and pathway analyzes are underway using Ingenuity Software to identify the biological functions and/or diseases that are most significant to the data set. The input data file contains gene annotations, p-values and fold change values developed using programs developed at MSU with R and SAS software. Pathways involving interferons and other cytokines as well as chemokines have been identified as critical for differentiating infected from vaccinated pigs. Final statistical analyses of the PRRSV study are still underway.