Confirmation of JEV infection in both <i>in vivo</i> and <i>in vitro</i> models using JEV specific antibody.

<p>The onset of JEV infection was confirmed by immunostaining of mouse brain sections and Neuro2a cells using JEV specific antibodies.</p

Validation of proteomic results using Immunostaining.

<p>Differential expression of selected proteins in neuron following JEV-infection in <i>in vivo</i> model. Images are representative of three independent experiments.</p

Temporal expression of selected proteins in mouse brain with disease progression.

<p>BALB/c mice of either sex were injected with 3×10⁵ p.f.u. of JE virus of strain GP78 through tail vein. The animals were then dissected at 1, 3, and 5 day post (JEV) infection (d.p.i) and brain was isolated for protein extraction. (<b>A</b>) Immunoblot showing the protein expression levels during JEV infectionas compared to that of mock-infected control mice. (<b>B</b>) Densitometric analysis of the differential protein expression. Data is representative of three independent experiments.</p

Relative fold change in differentially expressed proteins in brain tissue post-JEV infection.

<p>Spot intensities were normalized by total valid spot intensities and mean of values from duplicate analytical gels from four biological replicates were subjected to paired <i>t</i>-test analysis. Protein spots showing altered expression between control and experimental groups (|ratio|≥1.5, <i>p</i><0.05) were marked and excised. Dark grey bars indicate spots from mock infected control sample while light grey bars indicate JEV infected sample. * = p<0.05, # = p<0.01. Data represented are means ± SD of four independent experiments.</p

A schematic representation of the host response to JEV infection.

<p>Bioinformatic analysis of the proteins identified during the study was carried out to identify the various pathways and biological processes involved in the virus-host interaction. Based on the derived data, JEV pathogenesis network was constructed. Red tabs represent viral proteins and pink tabs represent the host proteins identified in this study.</p

Expression of selected proteins in human neuroblastoma (SHSY 5Y) cells after JEV infection.

<p>SHSY 5Y cells were either mock-infected or infected with JEV (4.4×10⁹ pfu/mL) at multiplicity of infection (MOI) of 5 for 1.5 h. The expression of different proteins after JEV infection as compared to mock-infected controls was analyzed by immunoblotting. Data represented are means ± SD of three independent experiments. Dark grey bars indicate spots from mock infected control sample while light grey bars indicate JEV infected sample * = p<0.05.</p

Differential protein expression in JEV infected Neuro2A cells.

<p>(<b>A</b>) A representative 2-DE gel of Neuroblastoma cell (Neuro2a) lysate from mock-infected and JEV infected cells. Equal amount of total protein from cell lysates were separated on an immobilized linear pH gradient IPG strips (4.0–7.0) and then in the second dimension on 10% SDS-PAGE. Spots showing differential expression were marked excised, and identified by MALDITOF/MS and database searches. The spots are labeled on the gel according to the numbers presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090211#pone.0090211.s007" target="_blank">table S1</a>. (<b>B</b>) Protein spots representing relative fold change in expression in mouse neuroblastoma cell (Neuro2a) lysate.Spot intensities were normalized by total valid spot intensities and mean of values from duplicate analytical gels from four biological replicates were subjected to paired <i>t</i>-test analysis. Protein spots showing altered expression between control and experimental groups (|ratio|≥1.5, <i>p</i><0.05) were marked and excised. Dark grey bars indicate spots from mock infected control sample while light grey bars indicate JEV infected sample. * = p<0.05. Data represented are means ± SD of four independent experiments.</p