Methods

Cell culture and ILTV infection
Cell culture reagents were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Chicken embryo lungs were isolated from 19 day old specific-pathogen free (SPF) chicken embryos (Charles River Laboratories, North Franklin, CT, USA). Lung tissues were homogenized and incubated in a 0.125% trypsin solution for 30 min at room temperature (25°C). Cells dissociated from lung tissues were suspended in a 1:1 ratio of mammary epithelial growth media (MEGM; Lonza, Rockland, ME, USA) and Dulbecco's Modified Eagle's Medium (DMEM, 0.45% glucose) plus 2% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine in 10 cm tissue culture dishes (Sarstedt Inc., Newton, NC, USA) pretreated with 0.5% gelatin in PBS to improve cell adhesion. Cultured cells were grown at 39°C containing 5% CO2 until cells reached confluent monolayers (2 to 4 days). The USDA reference strain of ILTV (National Veterinary Services Laboratories, Ames, IA, USA) was used to infect the chicken embryonic lung cells at a multiplicity of infection (m.o.i.) of 0.1. Infected cells were incubated at 37°C for 1 hr with rocking gently every 15 min. After the incubation, 10 ml of media, 1:1 MEGM/DMEM, were added to each culture dish, and the cells were incubated at 37°C in 5% CO2 for up to 7 days. This research was performed under the permitted protocol approved by both the Institutional Biosafety Committee (IBC; permit number: 10007) of University of Arkansas and the Animal and Plant Health Inspection Service (APHIS; permit number: 102743) of United States Department of Agriculture (USDA).

Total RNA extraction
Total RNA was extracted from uninfected- or ILTV infected chicken embryonic lung cells at 1, 3, 5, and 7 dpi using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions. Total RNA was treated with DNase I (New England BioLabs Inc., Ipswich, MA, USA), and RNA was re-purified by the TRIzol reagent. The quality of RNA was checked by fractionation on an agarose gel (data not shown).

Probe labeling and microarray hybridization
A two color labeling microarray system was used to compare uninfected- and ILTV infected embryonic lung cells at 1, 3, 5, and 7 dpi. Fluorescently labeled complementary RNA (cRNA) probes were generated by using the Two Color Microarray Quick Labeling kit (Agilent Technologies, Palo Alto, CA, USA) and following the manufacturer's instructions. RNA spike-in controls were used to adjust possible dye effects following manufacturer's instructions. The Spike-in controls represent two sets of ten synthesized RNA mixtures derived from the Adenovirus E1A transcriptome with different concentrations in each set [29]. These spike-in sets were mixed with either uninfected control or infected samples and co-hybridized to arrays. Briefly, 2 μg of total RNA were mixed with Spike-ins and converted to cDNA using reverse transcriptase and oligo dT primers in which T7 promoter sequences were added. T7 RNA polymerase was used for the synthesis and labeling of cRNA with either Cy3 dye for the uninfected control or Cy5 dye for the ILTV infected samples. The fluorescently labeled cRNA probes were purified using the Qiagen RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA), and the concentration, fluorescent intensities, and quality of labeled cRNA probes were determined using a Nano-drop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). An equal amount (825 ng) of Cy3 and Cy5 labeled cRNA probes were hybridized on a 4 × 44 K Agilent custom chicken oligo microarray (array ID: 017698). The hybridized slides were washed using a commercial kit package (Agilent Technologies, Palo Alto, CA, USA) and then scanned using a Genepix 4000B scanner (Molecular Devices Corporation, Sunnyvale, CA, USA) with the tolerance of saturation setting of 0.005%. Three biological replicates were conducted.

Microarray data collection and analysis
Background-corrected red and green intensities for each spot were used in the subsequent analysis. Global normalization based on local polynomial regression (loess) was applied to the intensities to remove effects that were due to undesirable systematic variations in microarray experiments rather than biological differences. The average values of the resulting normalized expression values in replicate hybridization sets were considered in the subsequent analysis. In order to identify a given set of genes that exhibited major alterations over time, a model-based clustering method [30] was employed, and the genes in the cluster were considered as differentially expressed over the time period. All analytic techniques were implemented in R (http://www.R-project.org).

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Reverse transcription was performed with 3 μg of total RNA using Superscript II reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA, USA) with oligo dT12-18 primers (Invitrogen Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions. The reverse-transcribed cDNA were diluted by 1:10 ratio and a portion (1 μl) of each product was subjected to qPCR under the following conditions: 40 cycles of 95°C for 30 s, gene-specific annealing temperature for 62°C for 1 min, extension for 30 s at 72°C, and a final extension at 72°C for 10 min. A non-template control and endogenous control (chicken GAPDH) were used for the relative quantification. The differential expression levels for the ILTV infected group were compared by the 2-ΔΔCT method against the uninfected controls [31]. Primers for qRT-PCR were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi) with these parameters: amplicon length, 95-100 bp; primer length, 18-27 nucleotides; primer melting temperature, 60-64°C; primer and amplicon GC content, 20-80%; difference in melting temperature between forward and reverse primers, 1-2°C. Primers were synthesized by Integrated DNA Technologies (Coralville, IA, USA). Primer information is listed in Table 2.

Bioinformatics
Functional interpretation of differentially expressed genes was analyzed in the context of gene ontology and molecular networks using the Ingenuity Pathways Analysis (IPA) 6.5 software (Ingenuity Systems®; http://www.ingenuity.com). The differentially expressed genes were categorized, compared to genetic categories in the IPA database, and ranked according to p-values [54]. The IPA analysis determined the subcategories within each category which is supplied with an appropriate p-value and the number of genes identified. Since the size of the created network could potentially be enormous, the IPA software limited the number of molecules in the network to 35, leaving only the most important ones based on the number of connections for each focus gene (focus genes = a subset of uploaded significant genes having direct interactions with other genes in the database) to other significant genes [55].