Construction and Rescue of a Minigenome System for Lentogenic D58 Strain of Avian orthoavulavirus 1 (Newcastle Disease Virus)

Abstract

Background: In reverse genetics studies, minigenomes of negative sense RNA viruses are used to model the process of virus replication and evaluate the virus rescue system in the course of construction of recombinant virus. In the current study, a minigenome (MG) rescue system for lentogenic D58 vaccine strain of Avian orthoavulavirus 1 (AOAV-1) (Newcastle disease virus) was developed to establish a reverse genetic system for this strain. Methods: The minigenome was constructed by assembling the trailer and leader regions of AOAV-1 D58 strain flanking the reporter gene, Aequorea coerulescens green fluorescence protein (AcGFP) in a modified transcription vector. Construction of this Minigenome was done using a simple restriction free technique-Splicing by Overlap Extension PCR. The constructed minigenome was evaluated for the reporter gene expression by transfection in T7 RNA polymerase expressing BSR/T7 cell line. Further, the replication and packaging ability of the constructed minigenome were also analyzed. Result: The reporter gene expression was confirmed in BSR/T7 cells which simulates the virus replication process and verifies the virus rescue system. In addition, this proves the applicability of restriction free cloning by Splicing by Overlap Extension (SOE) PCR for precise construction of the minigenome. The developed minigenome system can be used as a potential tool in the reverse genetic rescue of AOAV-1 D58 strain for the development of marker vaccine.