FIG 8.

The attenuation of rIBV M41-R is the result of substitutions C12137U (Pro85Leu) in nsp 10, G18114C (Val393Leu) in nsp 14, and G20139A (Val209Ile) in nsp 16. A series of rIBVs were generated which contained either C/U, G/C, U/A, or G/A at positions 12,137, 18,114, 19,047, and 20,139 within nsps 10, 14, 15, and 16, respectively. All rIBVs were generated through modification of the M41 cDNA in rVV M41-R to replace (rep) the nucleotides at positions 12,137, 18,114, 19,047, and 20,139 to resemble the pathogenic egg-derived M41-CK sequence (GenBank accession number MK728875.1). Eight-day-old SPF RIR chicks were inoculated with 10⁵ PFU of IBV. Each experiment included a pathogenic M41-CK-infected group as well as a mock-infected group. The rIBVs assessed in each experiment are as follows: experiment 1 (A through C), rIBV M41-R, rIBV M41R-nsp14.15.16rep, and rIBV M41R-nsp10rep; experiment 2 (D through F), rIBV M41-K, rIBV M41R-nsp10.15.16rep, rIBV M41R-nsp10.14.16rep, rIBV M41R-nsp10.14.15rep, and rIBV M41R-nsp10.15rep; experiment 3 (G through I), rIBV M41-K, rIBV M41R-nsp10.16rep, and rIBV M41R-nsp10.14rep. In each experiment, birds were observed for clinical signs from 3 to 7 dpi. (A, D, G) The average number of snicks per bird per minute and (B, E, H) the percentage of birds exhibiting rales per group were calculated. (C, F, I) Ciliary activities were measured from tracheas harvested from three randomly selected birds per group at 4 and 6 dpi. The average percentage for each group is displayed with error bars representing SEM. Statistical differences were analyzed using a two-way ANOVA with Tukey analysis for multiple comparisons. Statistical differences are indicated by * (P < 0.05), ** (P < 0.005), and *** (P < 0.0005).