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.