3.2  Safety

3.2.1  Pathogenicity of the recombinants in one-day-old SPF chickens
The earliest death was observed at 3 dpi in the QX-like strain rIBYZ group, which virus was highly pathogenic to one-day-old SPF chickens; the final mortality of this group was 63.3% (Fig. 3 A). The morbidity of the infected chickens in the rIBYZ group reached higher than 80%, and the diseased chickens exhibited respiratory symptoms (e.g., sneezing, coughing, as well as tracheal and bronchiolar rales), and the severe cases presented with additional signs of listlessness, huddling, and ruffled feathers. At necropsy, lesions were detected both in the respiratory and urinary system, including mucus, hyperemia, and hemorrhage in the trachea, as well as swelling and urate deposition in the kidney. Edema and congestion were observed in the lungs in 20% of cases (Fig. 3B). However, following inoculation with the rH120 or rH120-S1/YZ strains, the morbidity rates of the chickens was less than 20%, with moderate respiratory signs of coughing in some of the chickens. All of the infected chickens presented no visible lesions in the kidney and the survival rate was 100% through 14 dpi in both the rH120 and rH120-S1/YZ groups (Fig. 3A and B). No obvious clinical signs, gross lesions, or death attributable to IBV was observed in the control group.
Fig. 3 Pathogenicity of the rIBV rH120, rIBYZ, and rH120-S1/YZ strains. (A) Percent survival of SPF chickens infected at 1-day-of age during the 14-day observation period. No mortality was observed in rH120, rH120-S1/YZ, or the control groups. (B) Gross lesions on the lungs and kidneys of the infected chickens at 5 or 7 dpi.

3.2.2  Tissue pathology
For the chickens inoculated with the QX-like strain rIBYZ, lesions in the trachea at 5 dpi were characterized as cilia necrosis and loss, epithelial cell congestion, hemorrhage, and lymphoid infiltration (Fig. 4 C). In the bronchial and capillary lumen of rIBYZ-infected lungs, congestion, hemorrhage, as well as erythrocyte and inflammatory cell infiltration was observed in some severe cases from 7 to 10 dpi (Fig. 4G). Moreover, erythrocyte and lymphocyte infiltration, as well as tubular dilation was observed in the kidney of rIBYZ-inoculated chickens (Fig. 4K). Similar to the rH120 group, chickens inoculated with rH120-S1/YZ presented with mild pathology in the trachea at the early infection time points of 3, 5 dpi. Lesions in the trachea consisted of the loss of epithelial cells and cilia, decreased number of secretory cells, and thickening of the mucosa (Fig. 4B and D). In contrast, there were no lesions observed in the lungs and kidneys of rH120 and rH120-S1/YZ-infected chickens during the infection period (Fig. F, H, J, and L). No histopathological evidence of damage was detected in the bursa of all the groups and no IBV-associated lesions were found in the tissue samples of the control chickens (Fig. 4A, E, I, M–P).
Fig. 4 Histopathological changes in the trachea, lungs, kidneys, and bursa of SPF chickens infected with the recombinant rH120, rIBYZ, and rH120-S1/YZ strains at 1-day-of-age. Black arrows in the trachea indicate cilia and necrosis loss. Black arrows in the lungs indicate congestion and hemorrhage. Trachea samples were collected at 3, 5 dpi (A–D); lung and bursa samples were collected at 7, 10 dpi (E–H; M–P); and kidney samples were collected at 5, 7 dpi (I–L). Black arrows in kidneys indicate tubular dilation, congestion, and inflammation. Scale bar = 100 μm or 200 μm.

3.2.3  Growth in tracheal, lung, and kidney tissues
The time-dependent levels of viral replication in the different tissues determined by real-time RT-PCR presented in Fig. 5 are consistent with the tissue histopathology results. Compared to rH120 and rH120-S1/YZ groups, one day after inoculation with the QX-like strain, rIBYZ, the viral load in all organs began to rise rapidly, except at 5 dpi in the trachea. The viral load in tracheal samples in the rH120-S1/YZ group increased and peaked at 5 dpi. The level of viral RNA was significantly lower than the rH120 group both at 1 dpi and 7 dpi (Fig. 5A). The viral load in lung samples from all infection groups increased starting at 3 dpi, and the viral RNA in the lungs of the rH120-S1/YZ group was significantly lower compared with both the rH120 and rIBYZ groups (Fig. 5B). The viral load in the kidney of all IBV-inoculated groups was detected at 1 dpi. In contrast to the rH120 and rH120-S1/YZ groups, the viral growth in kidney of rIBYZ-infected chickens exhibited an extremely significant increase at 3 dpi and peaked at 5 dpi. The viral RNA copies of the rH120-S1/YZ group peaked at 1 dpi, after which they gradually decreased (Fig. 5C). No viral load was detected in the control samples at any of the time points.
Fig. 5 Viral load in the different organs at 1, 3, 5, and 7 dpi of SPF chickens infected with the recombinant strains rH120, rIBYZ, and rH120-S1/YZ at 1-day-of age. The bars indicate means ± standard deviations. ***Indicates an extremely significant difference at P ≤ 0.001; **Indicates a highly significant difference at 0.001 < P ≤ 0.01; *Indicates a significant difference at 0.01 < P ≤ 0.05.

3.2.4  Viral load in the oral and cloacal swabs
In all IBV infection groups, the viral load in both the oral and cloacal swabs generally showed a gradual decline from 7 dpi to 26 dpi. Compared with the rH120 group, The reduction in viral load between 14 or 18 dpi was significantly greater for chickens infected with rH120-S1/YZ than for chickens infected with rH120, and the viral RNA of the QX-like strain rIBYZ was lower than that of the rH120 group at 7, 14, and 18 dpi but with no statistically significance (Fig. 6 A). In contrast, the viral load in the cloacal swab of the rIBYZ group was significantly higher than that of the rH120 and rH120-S1/YZ groups at 7 dpi. Compared with other groups, the viral RNA in the cloacal swab of the rH120-S1/YZ group was significantly lower at 7, 14, and 22 dpi (Fig. 6B). No virus was detected in the control group at any time point.
Fig. 6 Viral loads at 7, 14, 18, 22, and 26 dpi in the oral and cloacal swabs of chickens infected with the recombinant rH120, rIBYZ, and rH120-S1/YZ strains. All data are presented as the mean ± standard error of the mean (SEM); ***Indicates an extremely significant difference at P ≤ 0.001; **Indicates a highly significant difference at 0.001 < P ≤ 0.01; *Indicates significant difference at 0.01 < P ≤ 0.05.