A reporter system for assaying influenza virus RNP functionality based on secreted Gaussia luciferase activity

Abstract

BACKGROUND: Influenza A virus can infect a wide variety of animal species including humans, pigs, birds and other species. Viral ribonucleoprotein (vRNP) was involved in genome replication, transcription and host adaptation. Currently, firefly luciferase (Fluc) reporter system was used in vRNP functional assay. However, its limitation for the testing by virus infection resulted in an increased need for rapid, sensitive, and biosafe techniques. Here, an influenza A virus UTR-driven gene reporter for vRNP assay based on secreted Gaussia luciferase (Gluc) activity was evaluated. RESULTS: By measuring Gluc levels in supernatants, reporter gene activity could be detected and quantitated after either reconstitution of influenza A virus polymerase complex or viral infection of 293T and A549 cells, respectively. As compared with Fluc reporter, Gluc-based reporter was heat-tolerant (65°C for 30 min) and produced 50-fold higher bioluminescent activity at 24 h posttransfection. Signals generated by Gluc reporter gene could be detected as early as 6 h post-infection and accumulated with time. Testing by viral infection, stronger signals were detected by Gluc reporter at a MOI of 0.001 than that of 1 and the effects of PB2-627K/E or amantadine on influenza vRNP activity were elucidated more effectively by the Gluc reporter system. CONCLUSIONS: This approach provided a rapid, sensitive, and biosafe assay of influenza vRNP function, particularly for the highly pathogenic avian influenza viruses.