Comparison of vRNA and cRNA based reporters for detection of influenza replication

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★ vRNA and cRNA based replicon reporters were designed for detection of influenza. ★ Reporters show strong correlation between influenza replication and luciferase signal. ★ vRNA based reporter was more sensitive in influenza detection. ★ Application of reporters to study kinetics of antiviral siRNA is demonstrated. ★ Possibility of secondary replication of progeny vRNA is suggested.

In this study, RNA polymerase I expressed replicons containing EGFP and luciferase reporter genes controlled by influenza vRNA or cRNA promoters were compared side-by-side in the ability to detect influenza RNA-dependent RNA polymerase activity as an indicator of influenza replication. Results showed the vRNA based Luc reporter was more sensitive to early detection of influenza virus at 6 h post infection (p < 0.05), and at 10-fold lower titer (MOI = 0.001). Lower sensitivity of cRNA based Luc reporter constructs was due to its background expression, 2-fold lower expression, and around 4 h delay in expression of luciferase. Despite these differences, both cRNA- and vRNA-based reporters demonstrated strong correlation between MOI and luciferase signal, and can be used for effective and early detection of influenza infection in vitro. Further, we demonstrated that these reporters can be used successfully to study the kinetics of antiviral drugs including siRNA. Our results also suggest that progeny vRNAs might participate not only in secondary transcription but also in secondary replication. The developed cRNA and vRNA reporters may help with further elucidation of the replication model of influenza A virus.

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