An amino acid insertion in the PHI loop of the VP2 capsid protein of serotype 1 infectious bursal disease virus deeply modifies its antigenicity and abrogates its propagation into B cells

Figure 1. Partial VP2 amino acid and nucleotide alignments of parent CT and mutant EM6 strains. A Amino acid alignment from VP2 amino acids 206 to 335 including the four loops PBC (hydrophilic peak A), PDE, PFG and PHI (hydrophilic peak B). B Nucleotide alignment (up) and amino acid translation (down) from nucleotide positions 1075 to 1128 encoding the PHI loop. The « GGG» nucleotides insertion encoding an additional Gly residue in EM6 is boxed. Numbers refer to the amino acid or the nucleotide positions in the segment A or polyprotein of the CT strain (GenBank accession number AJ310185). Dashes indicate amino acid or nucleotide identical to the parent CT strain. Asterisks identify conserved positions. The grey boxes indicate the loops.

Figure 2. Structure of a VP2 monomer in the absence or presence of a Glycine insertion at VP2 position 323. A Structure of a wild type VP2 monomer from a typical frame in the MD simulation with a closer view of the region of interest and a top view (boxed). B Structure of a VP2 monomer with the insertion of a Glycine at position 323 from a typical frame in the MD simulation with a closer view of the region of interest and a top view (boxed). Amino acids of interest are shown individually: 222 in orange, 223 in blue, 320 in purple, 321 in yellow and 323 insertion in red.

Figure 3. Growth kinetics of rCu1 and rCu1 ins[G323] viruses on primary CEF and chicken bursal cells. Replication kinetics (MOI = 3) of rCu1 and rCu1 ins[G323] on primary CEF (left panels) or primary chicken bursal B cells (right panels). Values corresponds to means, and error bars were associated (SD values). A Virus was titrated from cell-free supernatants harvested at the indicated time points. The limit of detection (dotted line) corresponds to 2.3 log10 TCID50/mL. B Quantification of B + strand by real-time RT-PCR from cells harvested at the indicated time points. The limit of detection (dotted line) corresponds to 2.6 log10 copies per 50 ng of total RNA.

Figure 4. VN titers of serum collected in rCu1 and rCu1 ins[G323] chicken groups in experiments #1 and #2. rCu1 (left panels) and rCu1 ins[G323] (right panels) were both used as antigen for VN tests on sera collected from experiment #1 at 22 dpi (A) and from experiment #2 at 20 dpi (B). Data are expressed in log2. The limit of detection (dotted line) corresponds to 3.32 log2. Each symbol represents a chicken. The median value is indicated by a bar. Sets of data were evaluated for significance using the non-parametric Kruskal–Wallis test with Dunn’s post hoc test (**p ≤ 0.01, ***p ≤ 0.001).

Figure 5. Bursal atrophy, histological lesions scoring of BF and viral RNA load into BF of rCu1 and rCu1 ins[G323] chicken groups in experiments #1 and #2. A Bursa to body weight ratio in rCu1 and rCu1 ins[G323] chicken groups (inoculated and contact birds) in experiment #1 (22 dpi, left panel) and #2 (20 dpi, right panel). B Histological lesions scoring of experiment #1 BFs collected at 4 dpi (inoculated birds, left panel) and 22 dpi (inoculated and contact birds, right panel) from the rCu1 and rCu1 ins[G323] chicken groups. C. Copy numbers of IBDV segment B positive strand in experiment #2 BFs collected at 4 and 10 dpi from Cu1 and rCu1 ins[G323] chicken groups (inoculated birds only). Each symbol represents a chicken. The median value is indicated by a bar. Sets of data were evaluated for significance using the non-parametric Kruskal–Wallis test with Dunns post hoc test (*p ≤ 0.05, **p ≤ 0.01).