Kinetics of vRNA, cRNA, and mRNA synthesis of NP and H5 genes in CEFs inoculated with rgDkYK10 and rgDkYK10-B5. CEFs were inoculated at an MOI of 10 with rgDkYK10 or rgDkYK10-B5, and total cellular RNA was harvested at 0, 2, 4, 6, and 8 h p.i. Three micrograms of total RNA per sample was reverse transcribed into cDNA using Primescript reverse transcriptase (Takara) and specific primers for mRNA (A and D), vRNA (B and E), or cRNA (C and F) of the NP (A to C) and H5 (D to F) genes. The amounts of cDNA were used to determine the RNA amounts by real-time PCR for the NP and H5 genes (49). The average threshold cycle value (40 − CT) of triple wells was determined, and the relative viral RNA concentration (40 − CT) at each point is shown after the values at 0 h p.i. of the virus strains were adjusted. *, significant difference (P < 0.05).