Total RNA was extracted using an RNeasy Micro kit and submitted to deoxyribonuclease (DNase) treatment (Qiagen), following the manufacturer’s instructions. RNA concentration and purity were evaluated by spectrophotometry (NanoDrop 2000c, Thermo Fisher Scientific). RNA (500 ng) was reverse-transcribed with both oligo dT and random primers using the PrimeScript RT Reagent Kit (Perfect Real Time, Takara) in a 10-μl reaction. Real-time PCRs were performed in duplicate using Takyon Rox SYBR MasterMix dTTP Blue (Eurogentec) on an Applied Biosystems QuantStudio 5 instrument. Transcripts were quantified using the following program: 3 min at 95°C followed by 35 cycles of 15 s at 95°C, 20 s at 60°C, and 20 s at 72°C. Values for each transcript were normalized to expression levels of RPL13A (60S ribosomal protein L13a) using the 2−ΔΔCt method. Primers used for quantification of transcripts by real-time qPCR were indicated as follows: RPL13A, 5′-AACAGCTCATGAGGCTACGG-3′ (forward) and 5′-TGGGTCTTGAGGACCTCTGT-3′ (reverse); IFN-α2, 5′-CTTGACTTGCAGCTGAGCAC-3′ (forward) and 5′- GCTCACCCATTTCAACCAGT-3′ (reverse); IFN-β, 5′-TGCTCTCCTGTTGTGCTTCTC-3′ (forward) and 5′-CAAGCCTCCCATTCAATTGCC-3′ (reverse); TRIM8, 5′-CTGCCGTGCAAACACAACTTC-3′ (forward) and 5′-TCCACGATGTTGGTGAGCTTC-3′ (reverse); Pin1, 5′-GAGAAGATCACCCGGACCAA-3′ (forward) and 5′-AAAGTCCTCCTCTCCCGACT-3′ (reverse).

RT-qPCR in zebrafish: Each point in Fig. 7 (D and H) corresponds to an individual larva. Expression levels of each transcript were normalized to expression levels of Ef1a. The following primer sequences were used, as previously described in (32): Ef1a, 5′-GCTGATCGTTGGAGTCAACA-3′ (forward) and 5′-ACAGACTTGACCTCAGTGGT-3′ (reverse); Ifnphi1 (secreted isoform), 5′-TGAGAACTCAAATGTGGACCT-3′ (forward) and 5′-GTCCTCCACCTTTGACTTGT-3′ (reverse); Ifnphi3, 5′-GAGGATCAGGTTACTGGTGT-3′ (forward) and 5′-GTTCATGATGCATGTGCTGTA-3′ (reverse); CHIKV-E1 (degenerate to be indifferent to silent mutations), 5′-AARTGYGCNGTNCAVTCNATG-3′ (forward) and 5′-CCNCCNGTDATYTTYTGNACCCA-3′ (reverse).

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