Attenuation and altered replication of IBV strain tl/CH/LDT3/03 after serial passage in chicken embryo fibroblasts and Vero cells

To adapt the infectious bronchitis virus (IBV) for cell culture, the tl/CH/LDT3/03 strain was subjected to serial passaging in chicken embryo fibroblasts (CEFs) and Vero cells, respectively. Cytopathic effects (CPEs) first became apparent at the 7th passage in CEFs and the 11th passage in Vero cells, respectively. The tl/CH/LDT3/03 strain achieved stable replication and adaptation after 20 passages in CEFs (CEA P20) and 25 passages in Vero cells (VEA P25). Analysis of the genomic sequences of the two adapted viruses identified amino acid substitutions, insertions, and deletions in some of the viral proteins. To evaluate the replication capacity of the cell-adapted viruses, 1-day-old SPF chicks were inoculated with either CEA P20 or VEA P25. Both CEA P20 and VEA P25 exhibited reduced replication capacity in chickens, as determined by viral titration in 11 selected tissues collected at 5 days post-inoculation (dpi). The pathogenicity of the two viruses was also decreased for 1-day-old chicks. Furthermore, VEA P25 elicited significantly reduced neutralizing antibody responses in infected birds, nearly 3-fold lower than that induced by CEA P20. To evaluate protective efficacy for chickens, in ovo vaccination of SPF eggs with either CEA P20 or VEA P25 was carried out. Both cell-adapted viruses provided complete protection against tl/CH/LDT3/03 challenge, therefore represent promising attenuated live vaccine candidates for in ovo vaccination. In conclusion, serial propagation of IBV tl/CH/LDT3/03 in CEFs and Vero cells resulted in successful viral adaptation, which was associated with decreased replication capacity and a consequent attenuation of virulence in chickens, showing the potential of live vaccine candidates against tl/CH/LDT3/03.