Avian infectious bronchitis virus disrupts the melanoma differentiation associated gene 5 (MDA5) signaling pathway by cleavage of the adaptor protein MAVS

Yu, Liping; Zhang, Xiaorong; Wu, Tianqi; Su, Jin; Wang, Yuyang; Wang, Yuexin; Ruan, Baoyang; Niu, Xiaosai; Wu, Yantao

To quantitate gene expression and IBV replication from IBV-infected CEK cells and chicken embryos, primers and probes specific for chMDA5 [23] , chIFN-β [24] , chIFN-λ, chMx [25] and IBV 5′-UTR (Table 1) were used for real-time PCR as previously described [26] . Briefly, RNA was extracted using an RNA extraction kit (MiniBEST Universal RNA Extraction Kit, Takara, China) according to the manufacturer's instructions. A total of 1 μg of RNA was then reverse transcribed to cDNA using a reverse transcription kit (HiScript Q RT SuperMix for qPCR, Vazyme, China) according to the manufacturer's instructions, after which the transcribed products were diluted and stored at −20°C. Gene expression was quantitated using a LightCycler 2.0 System (Roche Diagnostics Ltd., Switzerland). The relative expression ratios of the target genes chMDA5, chIFN-β, chIFN-λ and chMx were calculated using the △△Ct method. To assess IBV replication in ovo and in vitro, real-time PCR was performed by absolute quantitation PCR [26] .