Abstract
BackgroundInfluenza 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.ResultsBy 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.ConclusionsThis approach provided a rapid, sensitive, and biosafe assay of influenza vRNP function, particularly for the highly pathogenic avian influenza viruses.
Highlights
Influenza A virus can infect a wide variety of animal species including humans, pigs, birds and other species
Formation of a viral polymerase complex consisting of polymerase basic 2 protein (PB2), polymerase basic 1 protein (PB1), and polymerase acidic protein (PA) proteins together with NP is required for successful replication [10]
A mutation resulting in a change of lysine (K) to glutamic acid (E) was introduced into the PB2 gene of QH strain; this virus was named QH (PB2-627E)
Summary
Influenza A virus can infect a wide variety of animal species including humans, pigs, birds and other species. An influenza A virus UTR-driven gene reporter for vRNP assay based on secreted Gaussia luciferase (Gluc) activity was evaluated. Bioluminescence reporters such as firefly, click-beetle, Renilla, Gaussia, Cypridina luciferases, and calciumactivated photoproteins are widely used in molecular biology. Lutz and co-workers [11] introduced influenza A virus-inducible reporter gene segments (VIRGS, 3’ and 5’UTR sequences of the A/WSN/33 NP segment) into a Fluc reporter system to detect and quantify RNP activity. This system has been used to study influenza virus RNP activity by transfection or viral infection of susceptible cells
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