Construction & establishment of two minigenome rescue systems for Chandipura virus driven by recombinant vaccinia virus expressing T7 polymerase.

Abstract

Background & objectives:Chandipura virus (CHPV) is an emerging pathogenic rhabdovirus with a high case fatality rate. There are no reports of a minigenome system for CHPV, which could help its study without having to use the infectious agent. This study was, therefore, undertaken for the establishment of T7 polymerase-driven minigenome system for CHPV.Methods:The minigenome rescue system for CHPV consists of three helper plasmids expressing the nucleocapsid protein (N), phosphoprotein (P) and large protein (L) based on a recombinant vaccinia virus expressing bacteriophage T7 polymerase (vTF7-3). The minigenome construct is composed of a reporter gene, flanked by the non-coding regions of CHPV. Two minigenomes were constructed in an antigenome or complimentary sense, expressing luciferase or green fluorescent protein (GFP). The minigenome system was evaluated by co-transfection of the minigenome construct and three helper plasmids into CV-1 cells and analysis of the reporter gene activity.Results:All the helper proteins were expressed from the helper plasmids confirmed by Western blotting. Expression of reporter genes was observed from both the GFP and luciferase-based minigenomes. Green fluorescence could be visualized directly in live CV-1 cells. Luciferase activity was found to be significantly different from control.Interpretation & conclusions:The results showed that the helper plasmids provided all the necessary viral structural proteins required for the production of minigenome mRNA template, which in turn could rescue the expression of reporter genes. Thus, these minigenomes can be applied to mimic the manifestation of CHPV life cycle.