Replication of rDTMUV-DB2mut3 is attenuated in cultured vertebrate cells. DEFs (A) or BHK-21 cells (B) grown in 12-well plates were infected with 1,000 TCID50 of rDTMUV or rDTMUV-DB2mut3, while control cells were mock infected. The development of CPE was observed at 48 and 72 h p.i. using a Nikon 80i fluorescence microscope. (C) DEFs grown in 12-well plates were infected with recombinant viruses at 300 TCID50 per well. At the indicated time points, the supernatants were harvested, and the copy number of viral genomic RNA was determined using RT-qPCR. The data are presented as means plus SD (n = 3). *, P < 0.05; ****, P < 0.0001 (Student’s t test). (D) Groups of 9-day-old duck embryos (n = 7) were mock infected or infected with rDTMUV or rDTMUV-DB2mut3 by allantoic-cavity inoculation. The viruses were used at 100 TCID50 (top) or 1,000 TCID50 (bottom) per embryo. The duck embryos were incubated at 37°C and assessed for survival daily.