Intracranial Administration of P Gene siRNA Protects Mice from Lethal Chandipura Virus Encephalitis

Kumar, Satyendra; Arankalle, Vidya A.

Fluorescent microscopy and Western blotting confirmed expression of P protein fused with GFP. The molecular weight of the fusion protein was approximately ,61.0 Kda. The molecular weight of the GFP protein is 29.0 Kda and the molecular weight of bacterial expressed P protein is 32.5 Kda [27] . Confocal microscopy of HEK-293 cells transfected with pAcGFP1N1 and pAcGFP1N1-CHPV-P showed cytoplasmic localization of fusion protein P gene siRNA validation with pAcGFP1N1-CHPV-P and CHPV At 24 hr post transfection, cells transfected with both P gene siRNAs (P-1 to P-4) and target expressing plasmid pAcGFP1N1-CHPV-P showed that P-2 siRNA could reduce the GFP expression and P gene expression approximately by 80% (Fig 2a- c c) (p,0.05), whereas P-1, P-3 and P-4 were not very effective. With respect to CHPV, efficacy of P-2 siRNA in reducing replication was 2logs at 24 hr and 1log at 48hr (p,0.05) as evidenced by plaque assay and real time one step RT-PCR (Fig 2d) . A dose response experiment using 10-300 pmol of P-2 siRNA and pAcGFP1N1-CHPV-P showed 100 pmol as optimum concentration (Fig 2e) .