Fast type I interferon response protects astrocytes from flavivirus infection and virus-induced cytopathic effects

Lindqvist, Richard; Mundt, Filip; Gilthorpe, Jonathan D.; Wölfel, Silke; Gekara, Nelson O.; Kröger, Andrea; Överby, Anna K.

Gene set enrichment analysis was used to identify classes of genes overrepresented in the dataset. IFNAR −/− cells showed a negative enrichment compared to WT in immune pathways and interferon signaling, while the positively enriched gene sets were below our cut-off adjusted p value < 0.05 (Fig. 6 ). Both IFNαB/D and supernatant treatment lead to enrichment in "interferon signaling" and "interferon alpha beta signaling" (Fig. 6) . Differentially expressed genes between WT and the three samples were analyzed using IPA to uncover predicted activation of canonical pathways and disease functions. Pathways involved in viral recognition and antiviral signaling as well as functions involved in viability of leukocytes and antiviral and inflammatory responses were found to be downregulated in IFNAR −/− compared to WT astrocytes (Additional file 1: Tables S3 and S4), which can explain why the IFNAR −/− cells responded poorly after viral infection. IPA analysis revealed activation of pathways involved in innate immune signaling and viral recognition, antiviral and immune response after treatment with IFNαB/D and supernatant (Additional file 1: Table S3 and S4). To extend our analyses and investigate what might be responsible for these transcriptomic changes, with focus on the difference between the IFNαB/ D and the supernatant treatment, we performed an IPA upstream analysis, and a Connectivity Map (CMAP) analysis. Both of these analyses evaluate the relationship(s) that might have led to the pattern of transcript regulation that we found after treatment. In Fig. 7a , we have plotted all upstream regulators that are predicted by the IPA analysis to be either activated or inhibited after IFNαB/D or supernatant treatment. Upstream regulators that are predicted to be activated after both treatments include several interferon regulated factors (IRF3, IRF7, STAT1) as well as IFNAR and IFNγ as the most activated. Each dot in Fig. 7a represents a regulator e.g., (IFN alpha/beta) with a circle of markers of upregulated transcripts found in the dataset (Fig. 7b) . Interestingly, five upstream regulators were predicted to be activated specifically after supernatant treatment (IL5, CD38, KMT2D, HIF1A, UCP1, and JAK2; Fig. 7a, y-axis >2) . The CMAP database is an orthogonal bioinformatics tool that correlates specific geneoverexpression with their associated transcriptomic changes. Our transcriptional changes after IFNαB/D or supernatant treatments were compared to the CMAP database and the connectivity scores are compared to each other in Fig. 7c , and top scoring genes included IFNB1, IFNγ, and CD40. IFNγ came up in both gene set enrichment analyses, IPA and CMAP (Figs. 6, 7a, b) , suggesting that TBEV induces IFNγ after infection in astrocytes. However, no differences in IFNγ mRNA expression levels were detected after infection in astrocytes (data not shown).