The population structure of an embryo-attenuated infectious bronchitis virus (IBV) Arkansas (Ark) Delmarva Poultry Industry (DPI)-derived vaccine was characterized during serial passages in chicken embryo kidney (CEK) cells and after back-passage in embryonated chicken eggs (ECE) and in chickens. Both conventional and deep-sequencing results consistently showed population changes occurred during adaptation to CEK cells. Specifically, 13 amino acid (aa) positions seemed to be targets of selection when comparing the vaccine genome prior to and after seven passages in CEK (CEKp7). Amino acid changes occurred at four positions in the spike (S) gene and, at two positions in the S gene, large shifts in frequencies of aa encoding were observed. CEK adaptation shifted the virus population towards homogeneity in S. The changes achieved in the S1 gene in CEKp7 were maintained after a back-passage in ECE. Outside the S gene, aa changes at three positions and large shifts in frequencies at four positions were observed. Synonymous nucleotide changes and changes in noncoding regions of the genome were observed at eight genome positions. Inoculation of early CEK passages into chickens induced higher antibody levels and CEKp4 induced increased respiratory signs compared to CEKp7. From an applied perspective, the fact that CEK adaptation of embryo-attenuated Ark vaccines reduces population heterogeneity, and that changes do not revert after one replication cycle in ECE or in chickens, provides an opportunity to improve commercial ArkDPI-derived vaccines.

We previously demonstrated that adaptation of an embryo-attenuated infectious bronchitis virus (IBV) Arkansas Delmarva Poultry Industry (ArkDPI)-derived vaccine to chicken embryo kidney (CEK) cells shifted the virus population towards homogeneity in spike (S) and nonstructural protein genes. Moreover, the typical Ark vaccine subpopulations emerging in chickens vaccinated with commercial Ark vaccines were not detected in chickens vaccinated with the CEK-adapted virus. In this study, chickens vaccinated with a low dose (1.6 × 10(3) EID50/bird, where EID50 is 50% embryo infectious dose) of CEK-adapted Ark vaccine at 5 days of age showed a significant reduction of IBV RNA in lachrymal fluids and decreased incidence of IBV RNA detection in tracheal swabs 5 days after challenge compared to unvaccinated challenged chickens. In a second experiment, 5-day-old chickens were vaccinated with 10(4) or 10(5) EID50/chicken of CEK-adapted Ark vaccine, and protection was compared to chickens vaccinated with 10(5) EID50/chicken of the commercial ArkDPI-derived vaccine from which the CEK-adapted virus originated. All vaccinated chicken groups showed a significant reduction of respiratory signs and viral load 5 days after Ark virulent challenge compared to unvaccinated challenged controls. No viral subpopulations different from the challenge virus were detected in chickens vaccinated with CEK-Ark after challenge. In contrast, IBV S1 sequences differing from the predominant population in the challenge virus were detected in several chickens vaccinated with the commercial Ark attenuated vaccine. From an applied perspective, the CEK-adapted IBV ArkDPI-derived vaccine is an improved and effective vaccine candidate with which to protect chickens against virulent Ark-type strains.

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