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

In the clustering graph, adjacent sample groups are linked by an edge, the node color represents the average viral genomic RNA levels of each sample group. Edge length or distance between nodes in the graph does not recapitulate spatial closeness of samples. contrasting genes). In comparison to MERS-CoV SA 1, there was an earlier host response to MERS-CoV Eng 1 at 7 and 12 hpi. The accelerated Calu-3 response to MERS-CoV Eng 1 may be the result of the difference in kinetics of viral gRNA replication, with MERS-CoV Eng 1 more efficiently replicating at 3 and 7 hpi compared to MERS-CoV SA 1 ( Figure 1A) . Alternatively, the delay in the host response to MERS-CoV SA 1 may be due to the virus more efficiently evading innate immune responses that leads to enhanced viral replication compared to MERS-CoV Eng 1 (Additional file 2: Table S4 ). Between the two viruses there are amino acid differences in ORF4a and PLpro, viral proteins known to modulate the innate immune response during MERS infection [5] [6] [7] . We further examined host gene expression at the early time-points (3, 7 and 12 hpi) and found significant enrichment of genes associated with the STAT3 pathway. STAT3 pathway genes CDC25A, MYC, SOCS3 and SOCS4 were more strongly induced by MERS-CoV Eng 1 compared to MERS-CoV SA 1, particularly at 7 hpi ( Figure 3B ). Increased SOCS gene expression and decreased expression of PIM1 gene in response to MERS-CoV Eng1 indicated decreased STAT3 activity and possibly differential induction of apoptosis-related pathways. In a direct virus comparison, differential expression of pro-apoptotic BID, BAX, and BIM genes was observed at the early time-points and by 24 hpi there was extensive cytopathic effects caused by both viral infections that was likely the result of caspasedependent apoptosis, as previously shown for MERS infection [20] . Of the 4861 contrasting genes, 2653 genes were also DE against time-and data set-matched mocks. Hierarchical clustering of the 4861 DE genes resulted in distinct gene clusters, with striking expression pattern contrasts between MERS-CoV SA 1 and MERS-CoV Eng 1 at 18 and 24 hpi. As shown in Table 2 , functional analysis of the five most prominent clusters with contrasting gene expression revealed enrichment of integrin linked kinase (ILK) signaling and epithelial adherens junction signaling pathways, glutathione metabolism, and interferon and pro-inflammatory signaling pathways. Genes related to glutathione metabolism included GSTM1 and GSTM3, which were strongly downregulated in response to both viruses. ISGs, IFIT1 and IFIT3, were highly induced in response to both MERS-CoV Eng 1 and MERS-CoV SA 1, with pronounced early up-regulation specifically in response to MERS-CoV Eng 1. Genes associated with ILK signaling and epithelial adherens junction signaling pathways were strongly downregulated in response to MERS-CoV SA 1, whereas MERS-CoV Eng 1 predominantly up-regulated expression of these genes at the late time points (Additional file 3: Figure S1 ). Within the highly enriched pathways, cellular genes including PVRL1, RHOF and CREBBP with highest expression contrasts between the two infections were chosen for confirmation by qRT-PCR (Additional file 4: Figure S2 ). Pro-inflammatory CSF3 gene was found more highly induced by MERS-CoV SA 1 (log ratios in MERS-CoV SA1 and MERS-CoV Eng1 respectively: 2.2 and 0.5), whereas CCL5 gene was more highly induced by MERS-CoV Eng 1 (log ratios in MERS-CoV SA1 and MERS-CoV E