Nucleotide and Amino Acid Analyses of Unique Infectious Bronchitis Virus (IBV) Variants from Canadian Poultry Flocks with Drop in Egg Production

Figure 1. Phylogenetic tree including unique isolates T-62 and CL-61 (denoted without background color) and 114 reference strains of IBV S1 gene sequences. The design is constructed with the maximum likelihood method with 1000 bootstrap replicates in MEGA 11 software. The colors and design of the tree were performed with the iTol online tool.

Figure 2. Recombination analysis of the T-62 IBV isolate based on the whole genome sequence using the RDP5 software. In (a), T-62 serves as the query sequence, while the lines show different pairs of analysis for recombination: yellow (Iowa state, Conn vaccine), green (Iowa state, T-62), and purple (Conn vaccine, T-62). In (b), the recombinant tree is presented, with the red area representing T-62 IBV as the recombinant, the green area indicating the Iowa state isolates (IA1162/2020-MW) as the major parent, and the blue area showing the Conn vaccine strain as the minor parent. Grey lines indicate regions of recombination. Dark grey areas represent consensus sequences. Light grey areas highlight breakpoints, indicating specific points where recombination has occurred. The Shimodaira–Hasegawa tree topology test yielded a p-value of <0.0001, based on 100,000 bootstrap replicates.

Figure 3. Recombinant analysis of the T-62 IBV isolate based on the S1 gene sequence using the RDP5 software. In (a), T-62 IBV serves as the query sequence, while the lines show different pairs of analysis for recombination: yellow (Georgia GA/10216, Iowa state), green (Georgia GA/10216, T-62), and purple (Iowa state, T-62). In (b), the recombinant tree is presented, with the red area representing T-62 IBV as the recombinant, the green area indicating the Iowa state isolates (IA1162/2020-MW) as the major parent, and the blue area showing the Conn vaccine strain as the minor parent. Grey lines indicate regions of recombination. Dark grey areas represent consensus sequences. Light grey areas highlight breakpoints, indicating specific points where recombination has occurred. The Shimodaira–Hasegawa tree topology test yielded a p-value of <0.0001, based on 100,000 bootstrap replicates.

Figure 4. Recombinant analysis of the CL-61 isolate of the IBV based on the whole genome sequence using the RDP5 software. In (a), CL-61 serves as the query sequence, while the lines show different pairs of analysis for recombination: yellow (Conn vaccine, T-62), green (Conn vaccine, CL-61), and purple (T-62, CL-61). In (b), the recombinant tree is presented, with the red area representing CL-61 IBV as the recombinant, the green area indicating the Conn vaccine as the major parent, and the blue area showing T-62 as the minor parent. Grey lines indicate regions of recombination. Dark grey areas represent consensus sequences. Light grey areas highlight breakpoints, indicating specific points where recombination has occurred. The Shimodaira–Hasegawa tree topology test yielded a p-value of <0.0001, based on 100,000 bootstrap replicates. Recombination in S1 gene CL-61 strain of IBV.

Figure 5. Recombinant analysis of the CL-61 isolate of the IBV based on the S1 gene sequence using the RDP5 software. In (a), the CL-61 strain of the IBV serves as the query sequence, while the lines show different pairs of analysis for recombination: yellow (Mass vaccine, Conn vaccine), green (Mass vaccine, CL-61), and purple (Conn vaccine, CL-61). In (b), the recombinant tree is presented, with the red area representing CL-61 IBV as the recombinant, the green area indicating the Mass41 vaccine-like (unknown) strain as the major parent, and the blue area showing T-62 as the minor parent of CL-61. Grey lines indicate regions of recombination. Dark grey areas represent consensus sequences. White areas highlight breakpoints, indicating specific points where recombination has occurred. The Shimodaira–Hasegawa tree topology test yielded a p-value of <0.0001, based on 100,000 bootstrap replicates.

Figure 6. Pairwise alignment of the amino acid sequence of the S1 glycoprotein of the current IBV strains compared to the Conn (a) and Mass (b) vaccines. Amino acid changes in HVR 1 (amino acids 38–67) are indicated by the blue color, while the red and green colors refer to changes in the residues of HVR 2 (amino acids 91–141) and HVR 3 (amino acids 274–387), respectively.

Figure 7. Identification of amino acid changes in the receptor-binding domain of the S1 glycoprotein (a) that are essential for virus–respiratory tract binding. (b) Alignment of the S1 glycoprotein revealed residue mutations in the current IBV isolates compared to the M41 IBV strain at positions 38, 43, 63, and 69.

Figure 8. Schematic drawing of the entire S1 glycoprotein referring to the distribution of amino acids under negative selection (top) and episodic diversifying selection (bottom) in both IBV isolates. Top: orange circles show conserved amino acids under negative selection; green and black circles indicate unique residues under negative selection in the CL-61 and T-62 strains of the IBV, respectively. Bottom: orange circles depict conserved residues under episodic diversifying selection; green and black circles refer to distinct sites under episodic diversifying selection in the CL-61 and T-62 IBV isolates, respectively. S1-NTD: N-terminal domain of S1 glycoprotein; and S1-CTD: C-terminal domain of S1 glycoprotein. Homology modeling and verification of 3D structures of S1 glycoprotein.

Figure 9. Three-dimensional structures (a–d) and Ramachandran plots (e–h) of S1 glycoprotein. CL-61 (a,e); T-62 (b,f); DMV/1639 (c,g); and Mass/SES (d,h). The 3D constructs were built by the I-TASSER server and viewed in the PyMOL software, while the Ramachandran plots were generated by the Vadar server. Prediction of potential N-glycosylation sites in S1 glycoprotein.

Figure 10. Three-dimensional structure of the complete S1 glycoprotein, highlighting the N-glycosylation in CL-61 (a) and T-62 strains of IBV (b). The N-glycosylated amino acids (indicated by red spheres) were predicted by the NetNglyc server. These sites were distributed in different portions of the S1 glycoprotein including the RBD (shown by the blue color) and S1-CTD (highlighted by the golden yellow). Six N-glycosylation sites at S1-NTD (indicated by the red color) are essential for the binding to epithelial cells of trachea (c–e). All these critical sites were maintained in the S1-NTD of the Mass/SES strain of the IBV ((c); indicated by cyans), while three out of six were found in the IBV T-62 S1-NTD ((d); shown by cyan). The S1-NTD of the CL-61 IBV strain had some N-glycosylated residues close to the previous sites ((e); shown by magenta). The PDB files of the S1 glycoproteins were viewed in the PyMOL software. Molecular docking of S1 glycoprotein against sialic acid.