Understanding the molecular basis leading the virulence of vesicular stomatitis virus in pigs

Comparison of the transcriptiomic profile using microarray analysis betwween primary porcine macrophages infected with recombinant vesicular stomatitis viruses Overall design: Infections of recombinant vesicular stomatitis viruses (VSV) were carried out on primary swine porcine macrophages. Six well plates containing 1x10^7 cells per well were infected at MOI of 10 TCID50 with each virus and incubated for five hours at 37 °C under 5% of CO2. Infections were conducted on triplicates. As a mock-infected samples 3 wells were incubated in the same conditions with macrophage media. After incubation, total RNA was extracted and used in microarray analysis using DNA microarrays (Array design #:Agilent-069270; 4 X 44k per slide) manufactured by Agilent Technologies. Low input RNA two color labeling kit (Agilent Technologies) was used to lable cRNA. Total RNA isolated from mock-infection was labeled with Cy5 as universal control. All samples including mock-infection were labeled with Cy3. One Cy3 labled sample was cohybrided with the Cy5 labled control in one hybridization chamber. After hybridization and washing, the arrays were scanned with a GenPix 4000B and the images were saved as tiff files. The expression data were extracted with Genpix 7.0 software for statisitcal analysis using R with librarys(limma, MASS, statmod, splines). LOESS method was applied to normalize interarray variation. The pig gene sequences were mapped to human genes with BLAST and manual annotation. Annotated probes were mapped to human genes, and human gene Entrezid were used in bioinformatic analysis with The Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool.