Adaptive truncation of the S gene in IBV during chicken embryo passaging plays a crucial role in its attenuation.

Like all coronaviruses, infectious bronchitis virus, the causative agent of infectious bronchitis in chickens, exhibits a high mutation rate. Adaptive mutations that arise during the production of live attenuated vaccines against IBV often decrease virulence. The specific impact of these mutations on viral pathogenicity, however, has not been fully elucidated. In this study, we identified a mutation at the 3' end of the S gene in an IBV strain that was serially passaged in chicken embryos, and showed that this mutation resulted in a 9-aa truncation of the cytoplasmic tail (CT) of the S protein. This phenomenon of CT truncation has previously been observed in the production of attenuated vaccines against other coronaviruses such as the porcine epidemic diarrhea virus. We next discovered that the 9-aa truncation in the S protein CT resulted in the loss of the endoplasmic-reticulum-retention signal (KKSV). Rescue experiments with recombinant viruses confirmed that the deletion of the KKSV motif impaired the localization of the S protein to the endoplasmic-reticulum-Golgi intermediate compartment (ERGIC) and increased its expression on the cell surface. This significantly reduced the incorporation of the S protein into viral particles, impaired early subgenomic RNA and protein synthesis, and ultimately reduced viral invasion efficiency in CEK cells. In vivo experiments in chickens confirmed the reduced pathogenicity of the mutant IBV strains. Additionally, we showed that the adaptive mutation altered the TRS-B of ORF3 and impacted the transcriptional regulation of this gene. Our findings underscore the significance of this adaptive mutation in the attenuation of IBV infection and provide a novel strategy for the development of live attenuated IBV vaccines. Author summary: The administration of live attenuated vaccines is currently recognized as the most effective strategy for preventing infections caused by IBV. Understanding how mutations arising during vaccine production influence IBV virulence is crucial for developing more effective vaccines. In this study, we focused on a specific mutation in the S gene of IBV, which arose during viral propagation in chicken embryos. This mutation resulted in the truncation of the CT region of the S protein and concurrently eliminated a critical motif required for the retention of the protein at the viral assembly site. Similar S protein truncation has been observed in the production of attenuated vaccines against other coronaviruses, suggesting that the mutation identified in IBV in the present study was an adaptive mutation driven by natural selection pressure. Our findings underscore the value of targeting this adaptive mutation site as a strategy for reducing IBV virulence, and for the development of effective live attenuated vaccines against IBV and other coronaviruses. [ABSTRACT FROM AUTHOR]