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United States Department of Agriculture

Agricultural Research Service

Research Project: SWINE VIRAL DISEASES PATHOGENESIS AND IMMUNOLOGY Title: Gene Expression Profiling of Porcine Reproductive and Respiratory Syndrome Virus Infected Porcine Alveolar Macrophages

Authors
item Miller, Laura
item Harhay, Gregory
item Lager, Kelly
item Smith, Timothy
item Neill, John

Submitted to: Porcine Reproductive and Respiratory Syndrome International Symposium
Publication Type: Abstract Only
Publication Acceptance Date: November 1, 2007
Publication Date: November 30, 2007
Citation: Miller, L.C., Harhay, G.P., Lager, K.M., Smith, T.P., Neill, J.D. 2007. Gene expression profiling of PRRSV-infected porcine alveolar macrophages [abstract]. Porcine Reproductive and Respiratory Syndrome International Symposium. Paper No. 47.

Technical Abstract: Identifying specific pathways that associate with variation in PRRSV replication and macrophage function can lead to novel gene targets for the control of PRRSV infection. Serial Analysis of Gene Expression (SAGE) is a powerful technique that allows a detailed and profound quantitative and qualitative knowledge of gene expression profile, with out previous knowledge of the sequence of analyzed genes. Total cellular RNA was prepared from in vitro cultivated mock-infected and PRRSV strain VR-2332-infected porcine alveolar macrophages (PAM) at 0, 6, 12, 16 and 24 hours after infection, and subjected to SAGE analysis to obtain >100,000 tags per time point. Identified unique mRNA tags were analyzed for their identity and relative abundance. Tags were mapped to transcripts and genes by exact regular expression matching to sequences in GenBank, Harvard Gene Index, Pig Expression Database (Japan), and the USMARC EST databases. Tag abundance was corrected for sequencing error using R and sagenhaft. Relative abundance was calculated based upon the number of times a tag is represented in a given SAGE library. Examination of the SAGE data indicated that there were changes in gene expression occurring in the PRRSV-infected PAM. More than 400 unique tags with significantly altered expression levels were identified (p'0.01 with Bonferroni correction). The validity and kinetics of expression of SAGE identified genes were evaluated using real-time RT-PCR. Control genes that do not respond to PRRSV infection were also included in the analysis as internal controls. Differential expression of the most abundantly expressed tag, corresponding to the ferritin light chain gene, was confirmed by real-time RT-PCR. The increase in the ferritin L transcript abundance, as well as a cytochrome p450 and thioredoxin, was found in cell culture. The pro-inflammatory cytokines IL-1 alpha and CCL4 (MIP1 beta) declined in transcript abundance in accordance with the findings of Lopez-Fuertes et al. (2000). The transcripts encoding RNA helicase RHIV-1 and Mx1 were induced between 0 and 10 h post-PRRSV-infection in accordance with Zhang et al. (1999).

Last Modified: 8/29/2014
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