Fig 2.

In vitro and in vivo properties of the recovered wild-type NSW2011 viruses. (A) Plaque morphology in Vero cells at 4 days after infection. Cells were fixed with 4% formaldehyde and stained with 0.2% crystal violet. (B) 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 MAb 4G2. KUN, Kunjin virus. (C) Viral growth kinetics in Vero and A549 cells. Cells were infected at a multiplicity of infection (MOI) of 1, and culture supernatant was collected at the indicated time points. The viral titer was determined by plaque assay on Vero cells. Error bars represent the standard errors of the means of 2 samples. (D) Survival of 28-day-old CD-1 mice (n = 10) challenged intraperitoneally with 1,000 PFU of virus. Mice were monitored for 21 days and sacrificed when signs of encephalitis were evident. (E) Virulence in 4-day-old chickens (n = 8) following subcutaneous inoculation with ∼1,000 PFU of the indicated viruses. Infected chickens were bled daily for the first 7 days after infection, and virus titers in the serum were determined by plaque assays on Vero cells.