Bisp-W blocks JEV RNA replication.

<p>(A) Viral titers were determined by plaque assay of N2A cell culture supernatants (22 h pi) infected with JEV and treated with DMSO or 5 µM of Bisp-W at the indicated time points. Error bars represent Mean ± SEM. ** P<0.005, **** <0001, * <0.01, ** 0.096 and ** <0.001 for respective time points. (B) Viral titers were determined by plaque assay of N2A cell culture supernatants infected with JEV and treated with PBS or 20 µM minocycline and DMSO or 5 µM of Bisp-W as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002005#s2" target="_blank">materials and methods</a>. Error bars represent Mean ± SEM. *** P = 0.0002. N.D: Not Detected. (C) Western blot analysis of N2A lysates prepared from JEV-infected and DMSO or Bisp-W-treated cells. C- Capsid. β–actin is shown for loading control. (D) Viral titers were determined from supernatants (extracellular) and cell lysates (intracellular) at 22 h pi by plaque assay of samples from N2A cells infected with JEV and treated with 5 µM of Bisp-W at 1 h post-infection. ***P = 0003 and 0.00002 respectively. (E) Total RNA was isolated from N2A cells infected with JEV and treated with 5 µM Bisp-W at 1 h post-infection. JEV genome copy numbers were estimated by quantitative real time PCR normalized to GAPDH mRNA. ** P = 0.0001 by unpaired two-tailed t-test. Error bars represent Mean ± SEM. Data are representative of two or more experiments performed with three replicates.