Construction of three recombinant NDVs containing different forms of the S gene of Egyptian IBV variant strain EG/CU/4/2014. Schematic representation of different forms of chicken-codon-optimized IBV S gene into NDV strain LaSota antigenome cDNA using PmeI site between P and M genes to generate IBV vaccine candidates. In each transcriptional cassette, NDV gene-end (GE), intergenic sequence (IGS), gene-start (GS) and Kozak sequences were added upstream of the IBV S ORF. In rLaSota/wt.S, the full sequence of the IBV S gene without any modification (Blue box) was inserted. In rLaSota/S(Y1145A) + Fct12, the tyrosine residue present at position 1145 of the IBV S protein was changed into alanine (GCC nt 3453–3455) and the modified S gene was fused to the last 12 amino acids of NDV fusion protein cytoplasmic tail domain (Grey box). In rLaSota/SΔct + Fct12, ecto- and transmembrane domains of IBV S protein were fused with the last 12 amino acids of NDV fusion protein cytoplasmic tail domain.