qRT-PCR.

The synthesis of first-strand cDNA was carried out using oligonucleotide dT primers and a Maxima H Minus first-strand cDNA synthesis kit (Thermo, USA). The qRT-PCR was performed in a Bio-Rad CFX96 system by using SsoAdvanced Universal SYBR green supermix (Bio-Rad). The procedure included initial denaturation for 30 s at 95°C and 45 cycles of amplification (5 s at 95°C and 30 s at 60°C), followed by 10 s at 95°C to evaluate the melting curve. The 2^−ΔΔCT method (33) was used to analyze relative gene expression data. Double references, GAPDH ({"type":"entrez-nucleotide","attrs":{"text":"NM_002046.7","term_id":"1519316078","term_text":"NM_002046.7"}}NM_002046.7) and B2M ({"type":"entrez-nucleotide","attrs":{"text":"NM_004048.4","term_id":"1825761146","term_text":"NM_004048.4"}}NM_004048.4), were applied for calibration. The canonical sgmRNA sequence of ORF3 provided by our data was used to design qPCR primers for ORF3. The sense primer was located at the discontinuous fused region of its sgmRNA, and the antisense primer was located at the ORFs. All primers are shown in Table S1.