Fig 3.

In vitro and in vivo properties of NSW2011 mutant viruses generated by CPEC. (A) Western blot with anti-E antibodies. Lysates from viral culture supernatants were treated with PNGaseF (+) or mock treated (−). Samples were separated on 10% SDS-PAGE gels, transferred to nitrocellulose membrane, and probed with anti-E MAb 4G2. (B) Plaque morphology in Vero cells at 4 days after infection. Cells were fixed with 4% formaldehyde and stained with 0.2% crystal violet. (C) Viral growth kinetics in Vero and A549 cells. Cells were infected at an MOI of 1, and culture supernatant was collected at the indicated time points. Viral titers were determined by plaque assay on Vero cells. Error bars represent the standard errors of the means of 3 independent experiments. (D) Survival of 28-day-old CD-1 mice (n = 10) challenged intraperitoneally with 1,000 PFU of the indicated viruses. Mice were monitored for 21 days and sacrificed when signs of encephalitis were evident.