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

ng1 respectively: 0.71 and 2.2). Examination of the aforementioned contrasting genes showed that only a small number of those were already differentially expressed at the early time-points. For example, expression of RHOF gene in response to MERS-CoV Eng 1 was increased 4-fold relative to mock at 12 hpi, whereas MERS-CoV SA 1 did not induce RHOF gene expression at this timepoint. We therefore focused on differences in the host response mainly at the later time-points that had the highest number of contrasting genes. To some extent, this was also observed for IFN-α2 gene expression, which showed higher up-regulation in response to MERS-CoV SA 1 at 24 hpi, as confirmed by qRT-PCR (Additional file 4: Figure S2 ). In addition, IFN-λ1 and IFN-λ2 were DE relative to mock with high up-regulation at 18 and 24 hpi for both viruses. There were no virus-specific differences in expression of pro-inflammatory cytokines, IL-1α or TNFα-IP3, which were highly up-regulated in response to both MERS-CoV Eng 1 and MERS-CoV SA 1. The cell migrationpromoting factor, TNFα-IP2 [21] , was highly up-regulated for MERS-CoV Eng 1 alone (Additional file 4: Figure S2 ). Cytokines appear to be important for MERS infection. To explore potential mechanisms regulating cytokine activity in response to MERS-CoV, we designed a microarray experiment to analyze Calu-3 responses to various cytokine treatments and develop signatures that were then examined in the context of MERS-CoV-infected Calu-3 cells. Calu-3 cells were treated with either human recombinant interferon IFN-α, IFN-γ, TNF or IL-1α, and cell lysates collected at different time points posttreatment for microarray. We found 399 DE genes responsive to recombinant IFN-α and 261 DE genes responsive to recombinant IFN-γ, and as expected, the majority of these genes were up-regulated following stimulation [22] . In response to pro-inflammatory cytokine treatment, we found 76 DE genes responsive to recombinant TNF and 383 DE genes responsive to recombinant IL-1α, which were also differentially expressed in response to MERS-CoV SA 1 or MERS-CoV Eng 1. Many of these genes showed cytokine-specific expression for IFN-α (260 DE genes), IFN-γ (107 DE genes), and IL-1α (209 DE genes ( Figure 4A ). TNF induced the least number of DE genes compared to the other cytokines (8 DE genes) . Within the cytokine-stimulated genes we identified a set of 149 genes showing strong contrasts at late time points (18 and 24 hpi) between MERS-CoV SA 1 and MERS-CoV Eng 1 ( Figure 4B ).