2.5  Growth kinetics in chicken embryos
To examine the viral growth ability in chicken embryos, a real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) method was established. According to the sequences of IBV from GenBank, primers were designed based on conservative area in the 5′-UTR. The upstream primer was 5′-CCGTTGCTTGGGCTACCTAGT-3′, and the downstream primer was 5′-CGCCTACCGCTAGATGAACC-3′. The amplification product was cloned to pMD18-T vector (Takara) as a positive plasmid and its concentration was measured. A gradient dilution of 5 × 102–5 × 108 copies/μL of the plasmid was used as template for quantitation test. By plotting the cycle threshold (CT) values against the copies of the plasmid, the standard curve was generated.
18 10-day-old embryonated SPF chicken eggs (six eggs/group) were inoculated with rH120, rIBYZ, and rH120-S1/YZ at a dose of 107 viral RNA copies/100 μL, and used for growth curve experiments. The allantoic fluids were collected separately by syringe from the six inoculated embryonated eggs of each group at 12, 18, 24, 36, 48, 72, 96 h per inoculation. RNA was extracted from the allantoic fluids using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. cDNA was obtained by reverse transcription using a PrimeScript RT Master Mix Perfect Real Time Kit (TaKaRa, Otsu, Shiga, Japan). The viral copies were measured by absolute quantitative method of Real-time PCR, which was performed using SYBR® Premix Ex Taq™ II (TaKaRa, Otsu, Shiga, Japan) on an Applied Biosystems 7500 Fast Real-time PCR System. The standard curve was plotted against the log of the template copy number. All of the assays were run in triplicate and the copy number of each virus was calculated according to the standard curve.