A Comprehensive Roadmap Towards Generation of Influenza B Reporter Assay Using a Single DNA Polymerase Based Cloning of Reporter RNA Construct

Abstract

AbstractMini-genome reporter assay is a key tool for conducting RNA virus research. But, procedural complications and lack of adequate literature pose major challenge towards developing these assay systems. Here we present a novel yet generic and simple cloning strategy for construction of influenza B virus reporter RNA template and describe extensive standardization of the reporter RNP/ polymerase activity assay for monitoring viral RNA synthesis in infection free setting. Using this assay system, we, for the first time showed the effect of viral protein NS1 and host protein PKC-Delta upon influenza B virus RNA synthesis. Additionally, the assay system showed promising results in evaluating efficacy of antiviral drugs targeting viral RNA synthesis and virus propagation. Together, this work offers a detailed protocol for standardization of influenza virus mini-genome assay and an excellent tool for screening of host factors and antivirals in a fast, user friendly and high throughput manner.