Schematic model of gene regulation and three-dimensional structure prediction of the NCR of segment 8 of ISAV. A. The potential leaky scanning mechanism for gene expression of the second ORF in segment 8 of ISAV. The mRNA transcripts for segment 8 for two groups of isolates studied that differ in their Kozak sequence (see Table 1). Start codon are underscored. Critical nucleotides are in bold. Non-critical nucleotides are in bold and italics. B. Three-dimensional modeling of the folding of the vRNA and cRNA calculated at 15°C for intermediate- and high-pathogenicity isolates and the corresponding free energy (ΔG). The theoretical spatial distribution of the domains described in the 2D models is highlighted in color (in vRNA, purple and green represent denatured regions, and the ORF is represented in pink; in cRNA, purple, green, and pink represent the denatured regions, and the ORF is presented in light blue; in both models, the stems are represented in gray).