Genome Evolution of Two Genetically Homogeneous Infectious Bursal Disease Virus Strains During Passages in vitro and ex vivo in the Presence of a Mutagenic Nucleoside Analog

Figures

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  FIGURE 1. IBDV titer evolution during passages in vitro, ex vivo, and in vivo. (A) Layout of the serial passage strategy. For in vitro passages, a constant multiplicity of infection (MOI) of 0.01 and 0.1 was used in chicken B cells and DF-1 cells, respectively. For ex vivo and in vitro experiments, in chicken B cells and DF-1 cells, respectively, viral stocks from each passage were titrated in chicken B cells to adjust the infecting dose for the next passage. For in vivo experiments, five specific pathogen-free (SPF) chickens were inoculated with 105 TCID50/animal in 0.1 mL PBS of rCu-1-p0 by the intranasal route. The five Bursae of Fabricius (BF) were collected 4 days post-inoculation, pooled and titrated in chicken B cells to adjust the infecting dose for the next passage. For each condition, each replicate of the final passage was sequenced by NGS (RNA deep-sequencing). Virus progeny titers after each of the 12 passages in cells in the presence or the absence of 7DMA treatment (B–D) or rCu-1 progeny titers after each of the 5 passages in chickens (E). 7DMA c (triangle): control for constant activity of the drug 7DMA; DF-1 were infected with rCu-1-p0 in the presence of 7DMA in parallel…

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  FIGURE 2. Predictions of RNA secondary structures in the 3′UTR in IBDV genome. (A) The original 3′UTR sequence of segment B in rvv-p0 featuring in bigger bold number the mutated nucleotide position after passaging. (B) The 3′UTR sequence of segment B in rvv-p12, featuring in a dotted circle the nucleotide substitution. (C) The original 3′UTR sequence of segment A in rCu-1-p0 featuring in bigger bold numbers the mutated nucleotide positions after passaging. (D) The 3′UTR sequence of segment A in rCu-1/p12/B, featuring in a dotted circle the nucleotide substitution. (E,F) The 3′UTR sequence of segment A in rCu-1-p12/B/7DMA, featuring in dotted circles the nucleotide substitutions. In circles, conserved regions in 3′UTR sequences. Energy levels for each structure are given in kcal/mol in brackets.

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  FIGURE 3. Analysis of pathogenicity in SPF chickens infected with rvv-p0, rvv-p12, and rvv-p12/7DMA. (A) Mean symptomatic index between 1 and 14 day post-inoculation (d.p.i.) in chickens infected with rvv-p0, rvv-p12, or rvv-p12/7DMA. This index ranges from 0, “lack of signs”; 1, “typical IBD signs with ruffled feathers; motility is not reduced”; 2, “typical IBD signs with dehydration; motility slightly reduced”; to 3, “typical severe IBD signs (ethical endpoint) with prostration or death.” (B) Cumulated mortality rate at 21 d.p.i. in chickens infected with rvv, rvv-p12 or rvv-p12/7DMA. Different letters indicate a p-value < 0.05 between the different groups using logistic regression. NI, non-infected group.

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  FIGURE 4. Bursal-to-body weight ratio, spleen-to-body-weight ratio, chicken B cells counting, viral load in Bursa of Fabricius and viral shedding in chickens infected with rvv-p0, rvv-p12, or rvv-p12/7DMA. (A,B) Bursal-to-body weight [b/B] ratio (‰) at 4 (n = 5 chickens per group) and 21 days (at least 5 chickens per group) post-inoculation (d.p.i.). (C,D) Spleen-to-body-weight [s/B] ratio (‰) at 4 (n = 5 chickens per group) and 21 d.p.i. (at least 5 chickens per group). (E,F) Chicken B cell concentration in blood at 2 and 21 d.p.i. (n = 10 chickens per group except for the rvv-p0 group). (G) Virus titer at day 4 in Bursa of Fabricius [in log10 (TCID50/mL), n = 5 chickens per group]. For A–G, points represent individual values; boxplots indicate median and interquartile range. (H) Median viral shedding per group (in viral RNA copies/μL) at day 0–4, 7, 14, and 21 from cloacal swabs. For all graphs, different letters indicate a p-value < 0.05 between the different groups using Kruskal-Wallis test followed by Fisher’s least significant difference test with Holm adjustment method for multiple comparisons. NI, non-infected group.

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  FIGURE 5. IBDV capsid protein and polymerase structures. (A) Ribbon diagram of VP2. The known structure of capsid protein for the cell culture adapted Ct strain (Coulibaly et al., 2005) is shown. The base, shell and projection domains are colored in green, purple, and pink, respectively. Cyan sticks shown residues identified in neutralization escape mutants (position 222) and cell culture adaptation (positions 253, 279, 284, and 330). Position 270 is shown in yellow. (B) Ribbon diagram of VP1. The known structure of IBDV polymerase (Pan et al., 2007) is shown. The fingers, palm, and thumb domains are shown in purple, pink, and green, respectively. The active site located from position 401 to 403 is shown in rainbow color and position 329 in orange. The N- and C-terminal domains are shown in line and colored in clear blue and dark green, respectively.