Temperature-sensitive phenotype of the recombinant viruses. Six 10-day-old embryonated SPF chicken eggs were inoculated with 103 EID50 (0.2 ml/egg) of the recombinant viruses and incubated at 37°C and 41°C for 48 hours. Viral titers were determined by log10EID50/ml at 37°C. The detection limit is 0.7 log10EID50/ml. The statistical analysis was performed using Student’s t test (★★, p < 0.01; ★, p < 0.05).