Recombination with a vaccine strain drives the evolution of a more virulent phenotype in avian infectious bronchitis virus

Fig. 1. Identification of four IBV isolates. (A) Tissue and swab samples were inoculated into the allantoic cavity of 9-day-old SPF embryonated chicken eggs. After 3 days, the allantoic fluid was collected and tested by RT-PCR for Infectious Bronchitis Virus (IBV), Newcastle Disease Virus (NDV), Avian Influenza Virus (AIV), and Infectious Bursal Disease Virus (IBDV). Lanes 1, 2, 3, and 4 correspond to isolates SX/2407, SX/2408, SX/2409, and SX/2410, respectively. (B) The four isolates were inoculated into the allantoic cavity of 9-day-old SPF embryonated chicken eggs. Embryonic lesions, such as dwarfing or malformation, were observed 6 days post-inoculation. (C) Allantoic fluid containing each isolate was serially tenfold diluted in PBS and inoculated into the allantoic cavities of 9-day-old SPF embryonated chicken eggs. The 50% Embryo Infective Dose (EID₅₀) was calculated 6 days later using the Reed-Muench method.

Fig. 2. Phylogenetic analysis of four IBV isolates from North China. A phylogenetic tree was constructed for isolates SX/2407, SX/2408, SX/2409, and SX/2410 using 85 S1 gene sequences representing different genotypes, obtained from NCBI as reference sequences. The tree was generated using the maximum likelihood method in IQ-TREE with 1, 000 bootstrap replicates and subsequently visualized using ChiPlot (https://www.chiplot.online/). The IBV isolates obtained in this study are marked with red pentagrams, while vaccine strains are indicated by green triangles.

Fig. 3. Genomic characterization and recombination analysis of IBV. (A) Schematic diagram of the IBV genome. (B-D) Recombination analysis of the complete genome sequences of SX/2407 (B), SX/2409 (C), and SX/2410 (D) was performed using BootScan. The analysis was conducted with SimPlot v3.5.1, using a window size of 500 bp and a step size of 50 bp. The identified recombination sites correspond to the full genome map in (A).

Fig. 4. Clinical symptom scores and survival curves of chickens following infection with specific IBV strains. (A) Clinical scores of chickens infected with SX/2407, SX/2408, SX/2409, and SX/2410. Chickens inoculated with PBS served as the control group (n=10 per group). Only show significance between the clinical observation group and the control group. Statistical significance is indicated as follows: NS, not significant; a, P<0.05; b, P<0.01; c, p < 0.001. (B) Survival rates of 1-day-old SPF chickens infected with SX/2407, SX/2408, SX/2409, SX/2410, or PBS (control). Mortality was monitored for an observation period of 14 days. Only show the comparison results of SX/2408 and SX/2409. Statistical significance is indicated as follows: a, P<0.05.

Fig. 5. Gross lesions in tissues and tracheal ciliary activity scores of chickens infected with the four IBV strains. (A) In comparison to the PBS control group, chickens infected with SX/2407, SX/2408, SX/2409, and SX/2410 exhibited visible lesions in the lungs, trachea, and kidneys. (B) Assessment of tracheal ciliary activity in infected chickens at 3, 5, 7, and 9 days post-infection. Statistical significance is indicated as follows: c, p < 0.001.

Fig. 6. Histopathological changes in SPF chickens infected with the four IBV isolates. Tissue sections from the trachea (A-E), lungs (F-J), and kidneys (K-O) were fixed in 4% paraformaldehyde (PFA) and stained with hematoxylin and eosin (H&E) for pathological examination. Changes are indicated by colored arrows: loss and lodging of cilia (brown arrows), necrosis of epithelial cells (green arrows), lymphocyte infiltration (red arrows), focal indistinct structure of respiratory capillaries (black arrows), focal loose structure of respiratory capillaries (orange arrows), capillary hemorrhage (yellow arrows), and edema of renal tubular epithelium (blue arrows). No significant lesions were observed in the trachea (A), lungs (F), or kidneys (K) of the control group.

Fig. 7. Horizontal transmission capacity of the four IBV isolates in chickens. (A) Viral shedding from the oropharynx of directly inoculated chickens and their contact-exposed counterparts was measured at 3 and 9 days post-infection. (B) Viral shedding from the cloaca of directly inoculated chickens and their contact-exposed counterparts was measured at 3 and 9 days post-infection.

Fig. 8. Viral replication in various organs of SPF chickens following infection with the four IBV isolates. At 3, 5, 7, and 9 days post-infection, three infected chickens were randomly selected from each group. Samples of the trachea (A), lung (B), spleen (C), kidney (D), and liver (E) were collected. Tissues were homogenized, and nucleic acids were extracted to determine the viral load in each organ via RT-qPCR.