Genetic and Phenotypic Investigations of Viral Subpopulations Detected in Different Tissues of Laying Hens Following Infectious Bronchitis Virus Infection

Infectious bronchitis virus (IBV) commonly produces a range of genetic sequences during replication, particularly in the spike 1 (S1)-coding portion of the S gene, leading to distinct subpopulations within the broader viral population. It has been shown that certain microenvironments exert selective pressure on the S1-coding sequences and their encoded proteins, influencing the selection of viral subpopulations in these environments. In this study, high-throughput next-generation sequencing (NGS) was used to analyze the S1-coding sequences from tissues of the respiratory, digestive, renal, and reproductive systems of specific pathogen-free (SPF) laying hens. These tissues were collected nine days after infection with the California 1737/04 (CA1737/04) IBV strain, which is known to cause varying degrees of pathology in these tissues. Using a specific bioinformatics pipeline, 27 single nucleotide variants (SNVs) were detected in the S1-coding sequences derived from different tissues. These SNVs shaped multiple subpopulations (SP1-SP15), with SP1 being the core subpopulation present in all tissues, while others were tissue-specific. The IBV RNA loads in the tissues were negatively correlated with the number of SNVs or the Shannon entropy values, and phylogenetic analysis revealed a genetic divergence in the S1-coding sequences from certain tissues with lower viral RNA loads, particularly those from the trachea and ovary. Furthermore, the SNVs were associated with nonsynonymous mutations, primarily located in hypervariable region 2 (HVR 2) within the N-terminal domain of S1 (S1-NTD), except for those in SP7, which was exclusive to the trachea and contained changes in HVR 3 in the C-terminal domain of S1 (S1-CTD). Overall, this study adds to the existing knowledge about IBV evolution by highlighting the role of tissue-specific environments in shaping viral genetic diversity.