*2.7. Real-Time Quantitative PCR (RT-qPCR)*

Total RNAs were extracted from MDCK cells at 6, 12, or 24 hpi, using the RNAfast200 kit (Fastagen Biotech, Shanghai, China) according to the Manufacturer's instructions. Reverse transcription (RT) was carried out on 0.5 μg of total RNA by using PrimeScript RT reagent kit (Takara, Tokyo, Japan). Reverse transcription (RT) was conducted using specific oligonucleotides for vRNA (5- -AGC AAA AGC AGG-3- ), cRNA (5- -AGT AGA AAC AAG G-3- ), and mRNA [oligo(dT)]. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific primer (5- -GAA GAT GGT GAT GGG ATT TC-3- ) was also included in the RT reaction mixture for vRNA or cRNA analysis. The quantitative real-time PCR was performed in a 20-μL reaction mixture containing 50 nM forward and reverse primers (NP forward primer 5- -GAT TGG AAT TGG ACG AT-3- , reverse primer 5- -AGA GCA CCA TTC TCT CTA TT-3- ; M1 forward primer 5- -AAG ACC AAT CCT GTC ACC TCT GA-3- , reverse primer 5- -CAA AGC GTC TAC GCT GCA GTC C-3- ; M2 forward primer 5- - CCG AGG TCG AAA CGC CTA TC-3- , reverse primer 5- -CTT TGG CAC TCC TTC CGT AG-3- ; NS1 forward primer 5- -CTT CGC CGA GAT CAG AAA TC-3- , reverse primer 5- -TGG ACC AGT CCC TTG ACA TT-3- ; NS2 forward primer 5- -GTT GGC GAA ATT TCA CCA TTG CCT TCT CT-3- , reverse primer 5- -TTA AAT AAG CTG AAA TGA GAA AGT TCT-3- ), 1 × SYBR green master mix (Takara Biotech, Dalian, China), and various amounts of template. To quantify the changes in gene expression, the change in threshold cycle (ΔCT) method was used to calculate the relative changes normalized against the GAPDH gene (forward primer, 5- -AAT TCC ACG GCA CAG TCA AGG C-3- ; reverse primer, 5- -AAC ATA CTC AGC ACC AGC ATC ACC-3- ). The CT was defined as the cycle at which fluorescence was determined to be significantly greater than the background. The ratio of viral RNA to the internal control was normalized to the control level 0 h after infection, which was arbitrarily set equal to 1.0.
