Submitted to: Developments in Biologicals
Publication Type: Peer reviewed journal
Publication Acceptance Date: 2/14/2008
Publication Date: 7/1/2008
Citation: Miller, L.C., Harhay, G.P., Lager, K.M., Smith, T.P., Neill, J.D. 2008. Effect of porcine reproductive and respiratory syndrome virus on porcine alveolar macrophage function as determined using serial analysis of gene expression (SAGE). Developments in Biologicals. 132:169-174. Interpretive Summary: The pig respiratory virus, porcine reproductive and respiratory syndrome virus (PRRSV), causes highly significant losses to the swine industry worldwide. The ability of the virus to persist in its host shows that it has mechanisms to evade host immune response. 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 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. Examination of the SAGE data indicated that there were changes in gene expression occurring in the PRRSV-infected PAM.
Technical Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen of swine worldwide and causes considerable economic loss. The main target of infection is the porcine alveolar macrophage (PAM). Infection of PAM by PRRSV causes significant changes in their function by mechanisms that are not understood. We have employed Serial Analysis of Gene Expression (SAGE) to examine the global expression of genes in PRRSV-infected PAM. Total cellular RNA was prepared from in vitro mock-infected and PRRSV strain VR-2332-infected PAM at 0, 6, 12, 16 and 24 hours after infection, and subjected to SAGE analysis to obtain >100,000 tags per time point. These sequences were processed to account for sequencing error before generating tag:count lists. These lists were deposited into a modified Identitag database for mapping to porcine and PRRSV genes. Identified unique mRNA tags were analyzed for their identity and relative abundance. Examination of the SAGE data indicated that there were changes in gene expression occurring in the PRRSV-infected PAM over time post-infection. 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.