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1) Product Images from "Recombinant Modified Vaccinia Virus Ankara Generating Excess Early Double-Stranded RNA Transiently Activates Protein Kinase R and Triggers Enhanced Innate Immune Responses"
Article Title: Recombinant Modified Vaccinia Virus Ankara Generating Excess Early Double-Stranded RNA Transiently Activates Protein Kinase R and Triggers Enhanced Innate Immune Responses
Journal: Journal of Virology
Figure Legend Snippet: MVA recombinants expressing excess early dsRNA from neo or EGFP transgenes induce increased IFN-β expression. (A) Schematic representation of the two types of MVA recombinants generating excess early dsRNA either from two neo inserts (top) or from two EGFP inserts (bottom), each with the corresponding control and reference constructs. IGR, intergenic region. (B) Total RNA from murine BALB/3T3-A31 cells infected with the indicated viruses (MOI 10) or mock infected for 6 h was digested with RNase A/T1 (ssRNase digest) or RNase A/T1/V1 (ss+dsRNase digest) or not digested, and duplicate RT-qPCR quantification of total EGFP transcript (both sense and antisense) was performed as described in Materials and Methods. The mean of the fold induction values of EGFP or C7L transcripts over mock in undigested samples was set to 100%, and the mean percentage of the remaining qPCR signals after the indicated RNase digests was calculated for EGFP and C7L transcripts. Shown is one out two independent experiments. Where error bars are not visible, the standard error was negligible. (C) MEFs in 6-well plates were mock infected or infected with crude stocks of the indicated MVA recombinants at an MOI of 10 in duplicate. Fold induction of IFN-β mRNA over mock was determined by duplicate RT-qPCR per sample using total RNA isolated from cells at 6 h p.i. using a commercially available TaqMan assay (Life Technologies) for the murine IFN-β gene. Poly(I·C) was transfected using Fugene HD at 2 μg/well. 18S rRNA served as the endogenous control in all RT-qPCR analyses. Where error bars are not visible, the standard error was negligible. (D) IFN-β amounts in supernatants of MEF cultures infected in parallel to those shown in panel C were determined at 14 h p.i. by ELISA. (E) Murine A31 cells were either preincubated with 40 μg/ml of AraC for 1 h or left untreated and infected in duplicate at an MOI of 10 with the indicated MVAs with either 40 μg/ml AraC throughout infection or without AraC. Cells were harvested at 6 h p.i. for isolation of total RNA. Messenger RNAs for murine IFN-β and the late F17R VACV gene were quantified by qRT-PCR analysis as described above.
Techniques Used: Expressing, Construct, Infection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Isolation, TaqMan Assay, Transfection, Enzyme-linked Immunosorbent Assay