2.7.2  Tissue tropism of early viral infection in vivo
A total of 100 1-day-old SPF chickens were assigned to four groups (n = 25 per group). The birds were inoculated with 200 μL allantoic fluid containing 105 EID50 of the rH120, rIBYZ, or rH120-S1/YZ strains, respectively via eye drop and the intranasal route and 200 μL PBS per chick was administered to the control group (n = 25) via the same route. The tracheas, lungs, kidneys, and bursa from the 5 inoculated birds per group were harvested at 1, 3, 5, and 7 dpi, weighed, and collected into 1 mL PBS per sample and frozen at −80 °C. After grinding the samples, the viral RNA was extracted using TRIzol, and the cDNA was obtained by reverse transcription using a PrimeScript RT Master Mix Perfect Real Time Kit (TaKaRa, Otsu, Shiga, Japan). The viral RNA copies from each of the different samples were detected by real-time PCR as described above. All assays were run in triplicate and the copy number for each virus was calculated according to the standard curve.