Mechanisms of Tissue Susceptibility to FMDV Persistent Infection Based on Genes Differentially Expressed between Target and Non-target Tissues

Comparison of the transcriptiomic profiles using DNA microarray analysis betwween the pharyneal (target of FMDV acute and persistent infection) and lung (target of FMDV acute infection) tissues of cattle Overall design: Six adult Holstein steers were used in this study. Three steers were aerosol-inoculated with FMDV-A24-Cruzeiro and three were mock inoculated. All steers were euthanized at 72 hours post infection. Necropsies and tissue collections were performed immediately after euthanasia. Eight tissues including the dorsal soft palate (DSP), dorsal nasal pharynx (DNP), mid anterior lung (LNG), nasal turbinate epithelium (NTE), interdigital cleft skin (IDC), coronary band (CB), tongue epithelium (TONG) and the metacarpal skin (MCS) were collected from each animal. Total RNA was extracted from the tissues using Qiagen total RNA isolation kit and used in microarray analysis using DNA microarrays (Agilent array design ID: 019963; Design Format: IS-45220-4-V1 or 4 X 44k per slide). Low input RNA two color labeling kit (Agilent Technologies) was used to labeled cRNA. RNA isolated from MCS was labeled with Cy5 as universal control, all individual samples were labeled with Cy3. One Cy3 labeled sample was co-hybrided with the Cy5 labeled control in one hybridization chamber. After hybridization and washing, the arrays the arrays were scanned with a GenPix 4000B and the images were saved as tiff files. All procedures followed protocols provided by Agilent Technologies. The expression data were extracted with Genpix 7.0 software for statistical analysis using R with libraries (limma, MASS, statmod, splines). LOESS method was applied to normalize inter-array variation with a background subtraction of 15. The cow gene sequences were mapped to human genes with BLAST and manual annotation using information displayed on the UCSC Genome Browser.