Fig. 3

Schematic overview for the generation of the complete genome of recombinant Newcastle disease virus (rNDV) using Asis-Sal-Pac BioBrick strategy. The Iranian NDV isolates were subjected to RT-PCR. Six subgenomic fragments of L, HN, Fmut, M, P, and NP were cloned sequentially into the Asis-Sal-Pac-BioBrick vector, flanked on the upstream side by a T7 RNA polymerase promoter (T7 promoter) sequence and, on the downstream side by the hepatitis delta virus ribozyme sequence (H) followed by a T7 terminator sequence (T). To generate the attenuated recombinant NDV, we introduced and confirmed (by Sanger sequencing) mutations in the cleavage sequence of the wild-type F gene (here named Fm). The assembled genome on the vector was designed in accordance with the “rule of six” for NDV