Cytokine systems approach demonstrates differences in innate and pro-inflammatory host responses between genetically distinct MERS-CoV isolates

Selinger, Christian; Tisoncik-Go, Jennifer; Menachery, Vineet D; Agnihothram, Sudhakar; Law, G Lynn; Chang, Jean; Kelly, Sara M; Sova, Pavel; Baric, Ralph S; Katze, Michael G

RNA isolation from Calu-3 2B4 cells infected with MERS-CoV SA 1 and subsequent hybridization to Agilent 4 × 44K human HG arrays was previously reported in [11] . RNA isolation from Calu-3 2B4 cells infected with MERS-CoV Eng 1 was performed following the same protocol used for MERS-CoV SA 1-infected Calu-3 samples and used for subsequent hybridization to Agilent 8 × 60K human arrays. Calu-3 2B4 microarray experiments with an infectious clone recombinant SARS-CoV (icSARS-CoV) (AY278741) was reported in [18] . Using Agilent QC criteria we removed one 12 hpi replicate infected with MERS-CoV SA 1 and one 7 hpi replicate infected with MERS-CoV Eng 1. Due to the absence of mock samples at 24 hpi in the MERS-CoV Eng 1, we used the mock samples harvested at 18 hpi for the comparison at 24 hpi. In order to merge MERS-CoV Eng 1 and MERS-CoV SA1 data sets, we considered only probes that were mapped to gene names (Refseq IDs) in both arrays. To remove batch effects from the merged data sets we applied the R function ComBat [39] . The resulting data set was then quantile normalized. For each sample, a log2 fold change value was calculated as a difference between log2 normalized data for each sample and the average of log2 normalized data for time-and data set-matched mock-infected samples. The same procedure, but handled as separate data sets, was applied to the cytokine treatment microarray data sets.