E-244: A key neurovirulence factor located in the <i>ij</i> hairpin of the viral E glycoprotein.

<p>(<b>A</b>) RNA infectivity and replicability. BHK-21 cells were mock-transfected or transfected with RNAs transcribed from the parent or one of the 14 E-244 mutant cDNAs. RNA infectivity (in PFU/µg) at 4 dpt was estimated by infectious center assay, coupled with staining of cell monolayers using an α-JEV antiserum, and virus yield (in PFU/ml) at 22 hpt was determined by plaque titration on BHK-21 cells. (<b>B</b>) Viral growth. BHK-21 cells were infected at an MOI of 0.1 with the parent or one of the 14 E-244 mutant viruses. At the indicated time points, culture supernatants were used for virus titration on BHK-21 cells. (<b>C</b>) Neurovirulence. Groups of 3-week-old ICR mice (<i>n</i> = 10 per group) were infected IC with serial 10-fold dilutions of each virus stock, and the LD₅₀ values were determined. (<b>D</b>) Homology model. The predicted model of the E ectodomain of JEV SA₁₄-14-2 was built based on the crystal structure of the E ectodomain of WNV NY99 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004290#ppat.1004290-Nybakken1" target="_blank">[79]</a>, and the model was then fitted into the cryo-EM structure of WNV NY99 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004290#ppat.1004290-Mukhopadhyay1" target="_blank">[18]</a>. Illustrated is an icosahedral asymmetric unit of the three E monomers on the viral membrane. Highlighted in the inset is the critical residue Glu at E-244 in the <i>ij</i> hairpin adjacent to the fusion loop of the viral E DII.