Genome-Wide Analysis of Serial Passage of the Infectious Bronchitis Virus Reveals Evolutionary Dynamics Underlying...

Background/Objectives: Serial passage in embryonated eggs is widely used to attenuate the infectious bronchitis virus (IBV) for vaccine production; however, the evolutionary processes underlying attenuation and immunogenicity remain incompletely understood. Here, we analyzed genome-wide viral evolution during serial passages to investigate how mutations emerge, persist, are lost, or become fixed over time and how these dynamics relate to changes in pathogenicity and immunogenicity. Methods: Deep sequencing was performed on 11 representative serial passages (P2-P79) of the UY/11/CA/18 strain, including two derivative lineages: P7 VIR (virulent) and P53 VAC (attenuated and immunogenic). Results: This study identified an early adaptive phase characterized by a limited set of mutations potentially associated with genome replication, viral RNA processing, and virion assembly, including a key change in non-structural protein 14 and variants in M and 3c (E). This phase was followed by a broader expansion of the variant spectrum across replicase genes and delayed accumulation of Spike protein variants. Most Spike changes emerged during later passages and exhibited transient dynamics, and only a subset reached a high frequency after the establishment of early replicase- and structural-associated changes. Consistent with these dynamics, P7 VIR diverged before the late accumulation of Spike variants and retained a pathogenic phenotype, whereas P53 VAC diverged after the emergence of early high-frequency variants but before the extensive late-stage Spike variation observed in P79, which was associated with reduced immunogenicity. Conclusions: These findings support a multi-step model of IBV attenuation in which progressive filtering of genome-wide variation shapes distinct evolutionary outcomes during serial passages. This evolutionary framework provides insight into the relationship between attenuation and immunogenicity and may help guide the rational design of live attenuated vaccines.