Fig 1. Genome map of NDV Mexico/01/10 (wtMex) and in vitro characterization of its recombinant derivatives.

(A) Genome map of wtMex, with amino acid lengths indicated in italics above the map and location of each ORF (upper diagram) and strategy of construction of a complete antigenome cDNA encoding recombinant Mex (rMex), with unique restriction sites noted (lower diagram). (B) Modification of the F protein cleavage site of rMex from RRQKR/F to GRQGR/L, yielding rMex/ Las Fc. (C) For evaluating production of syncytia, DF1 cells in six-well plates were infected with rLaSota or each rMex mutant virus at a multiplicity of infection (MOI) of 0.01, incubated for 72 h in the presence chicken egg allantoic fluid as a source of added protease, and visualized by immunoperoxidase staining using antiserum against the N protein of NDV, with viral antigen stained red. (D) Western blot analysis of proteolytic cleavage of the F0 proteins of rLaSota and rMex mutant viruses in infected DF1 cells that were infected at an MOI of 0.1, incubated in the presence of added allantoic fluid, harvested 24 hpi, and visualized with anti-NDV rabbit antiserum. The positions of the precursor protein F₀ and the cleavage product F₁ are indicated. The relative levels of the F₀ and F₁ proteins in the Western blot experiment were measured by Bio-Rad Gel Image analysis, and the efficiency of cleavage was determined by dividing the amount of F₁ by the amount of F₁ plus F₀. Each bar represents mean and standard error of the mean of triplicate samples.